EP3007727A1 - Polydendrons - Google Patents

Abstract

A method of preparing a non-gelled branched vinyl polymer scaffold carrying dendrons, comprising the living or controlled polymerization of a monofunctional vinyl monomer and a difunctional vinyl monomer, using a dendron initiator and at least one further initiator.

Description

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The present, invention relates to nanomaterials, in particular nanoraaterials having hybrid structures comprising a branched vinyl polymer scaffold together with dendritic components. The present invention is particularly, though not exclusively, concerned with such hybrid materials from the perspective of medical applications, for example the carrying and delivering of drags and other medically useful materials, the enhancement of therapeutic and diagnostic properties, and improved or more efficient or cost-effective formulations.

Dendrimers have been extensively studied in this context, amongst many other contexts. The word "dendrimer" was coined in the early 1980s, following work on cascade chemistry and arborols, to describe polymers which contain dendrons. A "dendron" is a tree-like, repeatedly-branched, moiety. Thus, a dendron is a wedge- shaped dendritic fragment of a dendrimer. Typically, dendrimers have ordered, symmetrical architectures. A dendrimer comprises a core from which several dendrons branch outwards, to form a three-dimensional, usually spherical structure,

Dendrimers can be prepared by step-wise divergent or convergent growth. Divergent procedures start at the core of the dendrimer and grow outwards. Convergent procedures prepare dendrons first and then couple the dendrons together. In convergent procedures, the dendrons are typically coupled together at their focal points (i.e. at the base of the "tree", or the apex of the dendritic wedge) via chemically addressable groups.

For a nanomaterial to carry and deliver a drug or other biologically useful material, it is necessary for it to exhibit suitable properties in aqueous media and to have suitable domains to encapsulate the drug (which, for most drugs, need to be hydrophobic domains) and/or means of conjugating, bonding or otherwise associating with the drug. It is also advantageous for the nanomaterial to be able to carry a high

"payload" of drug. Dendrimers satisfy these requirements. Due to their repeatedly branched iterative nature, they are large compared to non-polymeric active molecules and contain a large number of surface groups, and can therefore encapsulate, and/or be conjugated to, a large amount of material Whilst they can be inade from all kinds of chemical building blocks, they commonly comprise organic chains which provide hydrophobic microenvironmenis for drugs or other organic molecules. At the same time they can be stable in aqueous media so that drags or other hydrophobic materials can be delivered within the body.

Whilst dendrimers have many interesting properties and promising features, they also have significant disadvantages. Dendrimer syntheses are lengthy and costly. The production of ideally branched structures requires multiple repeated steps of synthesis, purification and characterisation. Maintaining a 100% degree of branching generates complexity and takes time and requires very controlled reaction conditions. Even with high levels of successful recovery between steps, the compound effect after several steps means that the overall mass recovery suffers significantly. Whilst convergent methods are better than divergent methods from the viewpoint of ease and speed of procedure, they are still arduous, and other problems beset convergent methods, for example steric difficulties hindering coupling.

Geometric realities of iterative branching mean that the crowding constraints at the surface of the dendrimer sphere limit the size of the nanomaterials. Therefore dendrimers typically have a maximum size of about 1 Onm. This limits the amount of materia! they can cany.

Further description of dendrimers and their structures, preparation and applications, can be found in numerous articles including: S,M , Grayson and J.M. Frechet, Chen Rev. 2001, 101, 3819-3867; H, Frauenrath, Prog. Polym. Sd 2005, 325-384; F. Aulenta, W, Hayes and S. Ramiard, European Polymer Journal 2Θ03, 39, 1741-

1771 : E.R. Gillies and J.MJ. Frechet, Drug Discovery Today, 21105, 10, 1 , 35-43: and 5,1-L Medina and M.E.H. El-Sayed, Chem. Rev, 2009, 109, 3141 -3157. From a first aspect the present invention provides a method of preparing a non-gelled branched vinyl polymer scaffold carrying dendrons, comprising the living or controlled polymerization of a monofunctional vinyl monomer and a difunctional vinyl monomer, using more than one initiator, at least one of which is a dendron initiator.

From a second aspect the present invention provides a non-gelled hranched vinyl polymer scaffold carrying more than one type of moiety, at least one of which is a. dendron moiety.

Thus the present invention provides products which can be referred to as

"poiydendrons" because they contain a plurality of dendrons. The dendrons may be the same or different. Poiydendrons retain the advantages of dendrimers without having their disadvantages of cost, complexity and arduous synthesis, instead of the dendritic structure extending all the way to the centre, the core is a tuneable arid cost- effective non-gelled branched vinyl polymer scaffold. The poiydendrons typically take the form of units (which optionally are approximately spherical) with a large number of external surface dendron groups and with the vinyl scaffolds typically being present predominantly in the centre of the units.

The non-gelied branched vinyl polymer scaffolds of the present invention exhibit good solubility and low viscosity. They can be contrasted with polymer structures which are insoluble and or exhibit high viscosity, such as extensively crosslinked insoluble polymer networks, high molecular weight linear polymers, or microgels.

The products can be made by, but are not limited to being made by, living polymerization, controlled polymerization or chain-growth polymerization. Several types of living and controlled polymerization are known in the art and suitable for use in the present in vention. A preferred type of living polymerization is Atom Transfer Radical Polymerization (AT P), however other techniques such as Reversible Addition-Fragmentation chain-Transfer (RAFT) and Nitroxide Mediated Polymerisation (NMP) or conventional free-radical polymerization controlled by the deliberate addition of chain-transfer agents are also suitable syntheses.

The skilled person is aware of techniques to provide branched but non-gelied vinyl polymer scaffolds. For example, suitable procedures are described in WO 2009/122220; N. O'Brien, A. McKee, D.C. Sherrington, A.T, Slark and A. Titterton, Polymer 2000, ¥i, 6027-6031 ; T. He, D.J. Adams, M.F. Butler, C.T. Yeoh, A.L Cooper and S.P. Rannard, Angew. Ckem. Int Ed. 2007, 46, 9243-9247; V. Butun, I. Bannister, N.C, Billingham, D.C. Sherrington and S.P. Amies, Macromolecules

2005, 38, 4977-4982; L Bannister, N.C. Billingham, S.P. Armes, S.P. Rannard and P. Findiay, Macromolecules 2006, 39, 7483-7492; and R.A. Slater, T.O McDonald, D.J. Adams, E.R. Draper, J.V.M. Weaver and S. P. Rannard, Sqfi Matter 2012,

9816-9827. The non-gelled and soluble products of the present invention are different to materials disclosed in LA. Connal, II. Vestberg, CJ. Hawker and G.G. Qiao, Macromolecules 2007, 40, 7855- 7863 which comprise multiple cross-linking in a gelled network.

The polymerization of each vinyl polymer chain starts at an initiator. Polymerization of monofunctional vinyl monomers leads to linear polymer chains.

Copolymerization with difunctionai vinyl monomers leads to branching between the chains. In order to control branching and prevent gelation there should be less than one effective brancher (difunctionai vinyl monomer) per chain. Under certain conditions, this can be achieved by using a molar ratio of brancher to initiator of less than one: this assumes that the monomer (i.e. the monofunctional vinyl monomer) and the brancher (i.e. the difunctionai vinyl monomer) have the same reactivity, that there is no intramolecular reaction, that the two functionalities of the brancher have the same reactivity, and that reactivity remains the same even after part-reaction. Of course, the systems and conditions may be different, but the skilled person understands how to control the reaction and determine without undue

experimentation how a non-gelled structure may be achieved. For example, under dilute conditions some branchers form intramolecular cycles which limit the number of branchers that branch between chains even if the molar ratio of brancher to initiator (i.e. polymer chain) is higher than 1 : 1 in the reaction. in the present invention, dendrons are used as macromolecular initiators. In order to be able to initiate polymerization, the dendrons must bear suitable reactive functionality. For example, in ATRP, convenient and effective initiators include alky! halides (e.g. alkyl bromides), and so dendrons which carry halides at their focal points can act as initiators, in this scenario, propagation starts at the apex of the dendron "wedge". The skilled person is well aware of the types of components and reagents which are used in ATRP and other living or controlled polymerizations, and hence the type of functionality which must be present on or introduced to dendrons for them to act as initiators,

One possible way of introducing bromo groups to dendrons is to functionalize dendron alcohols with alpha-bromoisobutyryl bromide. There are however many other ways of functionalizing dendrons so that they can act as initiators and other types of functionality which will initiate polymerization. The concept of a dendron initiator is applicable to all suitable types of polymerization and the functionality can be varied as necessary.

There is no particular limitation regarding the type of dendron that can be used, or the chemistry used to prepare the dendrons. in some scenarios it is desirable to have particular groups present at the surface (i.e. at the tips of the "branches" of the dendron), and these may be incorporated during the synthesis of the dendron. The dendrons are preferably non-vinyl. Any suitable coupling chemistry may be used to build up the dendrons. in one example, amines and alcohols may be coupled together, for example using carbonyldiimidazole. This is, however, merely one example and numerous other coupling methods are possible. If exclusively one type of dendron initiator were used then in the resultant hybrid branched product one end of each vinyl polymer chain would bear that dendron.

In contrast, an essential feature of the present invention is that mixed initiators are used, in other words not only a dendron initiator but also at least one further initiator (which may be a different type of dendron initiator, or alternatively an initator other than a dendron initiator) is used. This allows considerable advantages in terms of varying the composition and the properties of the resultant polydendron structure. The present invention resides in the combination of features which work well together. The branched vinyl polymer methodology is intermingled with the use of mixed initiators including at least one dendron initiator. The way in which the living or controlled polymerization occurs means that the different initiators are distributed statistically and evenly around the surface of the non-gelled branched vinyl polymer scaffold. Some polymer chains will have one type of initiator at one end whereas other polymer chains will have another type at their end. There may be two types of initiator, or more, e.g. three or four or more, and therefore the multiplicity of types of end group may be two or more.

The vinyl polymer core is easily tuneable and very cost-effective. Different types of monomers, with different properties (e.g. differing solubility properties) may be used. The methodology allows a sizeable scaffold to be built, and the molecular weight and size can be controlled by choice of particular monomers (a wide range can be used) and reaction conditions, for example the ratio of initiator to monomer. The material is non-gelled and therefore soluble. At the same time the use of mixed initiators allows further tuneability and flexibility. There are synergistic advantages: for example the use of dendrons and other moieties as initiators means that they do not need to be introduced separately but instead are used as reagents within an already very efficient and convenient polymerization process. The process conveniently and cost-effectively results in the different types of initiators being distributed throughout the materials. The initiators themselves are relatively easy to synthesize. Regarding the need for the initiators to have suitable means and functionality to initiate polymerization, the considerations described above in relation to the dendron initiators apply mutatis mutandis to the at least one further initiator(s).

The living or controlled polymerization methodology inherently allows control in the synthesis of the polymeric scaffold. For example, ATRP and other techniques are robust and flexible in being suitable for use with a large variety of functional groups and in avoiding unwanted side reactions. The size and dispersity of the products can be controlled. The monomer units are usually homogeneously distributed between the initiator molecules and therefore the chain length, and hence the molecular weight, can be controlled. The conditions can be controlled to result in materials having low pol dispersity indexes when forming linear polymers, i.e. mixtures wherein the individual components have approximately the same size. This is particularly useful in the present invention as the individual chains comprising the branched structure (i.e. the primary chains) have similar chain lengths. The resulting branched polymers of the invention have a distribution of structures with varying numbers of linear chains connected to form the branched architectures.

The use of at least one further initiator, in addition to the dendron initiator, within the living or controlled polymerization methodology, brings further advantages. The further initiator alters the properties of the polydendron, for example the solubility, hydrophilicity, hydrophobicity, aggregation, size, reactivity, stability, degradability, therapeutic, diagnostic, biological transport, plasma residence time, cell interaction, drug compatibility, stimulus response, targeting and/or imaging characteristics.

The further initiator may comprise or be derived from one or more of the following: a small molecule, a drug, an active pharmaceutical ingredient, a polymer, a peptide, a sugar, a dendron, a moiety which carries or can carry a drug, an anionic functional group, a cationic functional group, a moiety which enhances solubility (for example, of the polydendron within aqueous systems, or of a drug or other carried material), a moiety which prolongs residence time within the body, a moiety which enhances stability of a drug or other active material, a moiety which reduces macrophage uptake, a moiety which enhances controlled release, a moiety which enhances drug transport, or a moiety which enhances drug targeting.

The initiator may be a macroinitiator, for example a macroinitiator prepared by synthesis from one or more monomer (e.g. a water soluble monofunctional monomer), or a macroinitiator prepared by modification of a pre-syuthesized polymer. The macroinitiator may be a copolymer, i.e. may comprise a polymer made from at least two monomers, e.g. monofunctional monomers. The macroinitiator may further he selected from natural polymers, for example water soluble or partially soluble polymers, e.g. polysaccharides, polypeptides or proteins. Each type of initiator may fail within one or more than one of the above definitions; for example the initiator may he a dendron and may also carry a drag. The initiator may also be a pro-drug, releasing a moiety that becorn.es pharmacologically active fter farther process within the body.

The present inventors have been surprised at how effective the use of mixed initiators is, in allowing a range of properties to be controlled and tuned. As described in more detail below, they have observed: that the surface chemistry can be varied widely across a hydrophobic - amphophilic - hydrophilic spectrum; that the encapsulation environment can be varied significantly; that the salt stability can be controlled; and that transcellu!ar permeability (in an in vitro model) can be tuned and improved.

In view of the drag delivery capabilities, from further aspects the present invention also provides pharmaceutical compositions comprising the products of the present invention, and allows enhancements in terms of medical administration possibilities,

For example, the surprisingly effective way in which the polydendrons interact controllably with, and transport encapsulated materials through, model gut- epithelium, is relevant to oral delivery applications. Materials of this type are also useful within parenteral administration such as intravenous, subcutaneous and intramuscular injection.

Polyethylene glycol (PEG) groups are advantageous for use in the initiators of the present invention. In comparison to polydendrons which carry dendrons alone, polydendrons which carry not only dendrons but also PEG groups exhibit enhanced stability in aqueous systems, controlled interaction with cells, and prolonged systemic half-life. Non-limiting examples of suitable PEGs include those with end functionality such as methyl, hydroxyl, amine, acid etc, functionality, and/or those with molecular weights above 300 g mol, preferably those with hydroxyl and acid functional chains and/or with molecular weights >750 g/mol. Particularly preferred are hydroxyl compounds and/or those with molecular weights >1000 g/mol.

Alternatively, other chemical moieties which function in the same or similar way and which can advantageously be used in the present invention include acryiate and methacrylate moieties including water-soluble polymeric chains (e.g. less than 20000 g/mol), for example derived from vinyl or non vinyl monomers such as ethylene glycol methacylate, glycerol methacrylate, vinyl alcohol, acrylic acid, methacrylic acid, or hydroxyethyl methacrylate.

The initiators may include groups which allow post-functionaiization of the polydendrons. Thus, whilst various possible initiator structures and moieties have been discussed above, an alternative to them being present within the initiator at the start of the reactio is to incorporate them later by reaction of the polydendron with. suitable materials.

Suitable functional groups in initiators which allow post-fonctiona ization include thiols, hydroxy! groups, amines, acids or isocyanates, amongst others. For example, N-hydroxysuceimmide functionaiized initiators can be incorporated into polydendrons and post-tunctionalized with materials containing amine groups.

The several means of flexibility and levels of control provided by the present invention reside in the ability to alter several variables including: the amount of initiator(s) relative to vinyl polymer, the ratio between dendron initiator(s) and non- dendron initiators) [or other dendron initiators)], the nature and properties of the dendron initiato s), &e nature and properties of the non-dendron initiators), the extent of branching, the nature and properties of the monomerfs), the nature and properties of the hra.neher(s), and the capacity of the nanomaterials for drugs or other materials,

A further advantage of the methods and products of the present invention is that they are compatible with the preparation of nanomaterials which are stable and of controllable and uniform size. Nanoprecipitation of branched vinyl polymers is disclosed in R.A. Slater, T.O McDonald, D.J. Adams, E.R. Draper, J.V.M Weaver and S. P. Rannard, Soft Matter 2012, 8, 9816-9827. This technique has been successfully used on single and mixed initiator - carrying polydendrons of the present invention to prepare stable nanoparticles. The nanoparticles are prepared by self assembly during precipitation with the dispersity and size of these nanoparticies being effectively controlled by varying the nature of the solvents, precipitation method, concentration, and presence of other components. Uniform or near uniform assembled nanoparticle sizes with low polydispersities can be achieved.

Nanoparticies of uniform and controllable size are extremely useful in the field of drug encapsulation and delivery.

The nanoparticies may for example be prepared by precipitation of the polydendron out of solution using a solvent which is a non-solvent for the vinyl polymer scaffold but which is a good solvent for the dendrons or other surface groups,

This nanoprecipitation using a solvent switch might have been expected to lead to collapse of the internal vinyl polymer core, but self-assembly of the individual polydendron particles is observed leading to very stable distributions of larger complex nanoparticies with a narrow size distribution.

A preferred "non-solvent" for the vinyl polymer, i.e. medium in which the nanoprecipitate particles are stable, is water.

By way of example, where the core is a poiyHPMA-EGDMA material and the dendrons are selected from Gl or G2 (shown in Figure 1), then the material can be first dissolved in THF and nanoprecipitated into water , or first dissolved in acetone and then precipitated by adding hexane.

The characteristics of the polydendron, including the electronic/ charge and steric nature, and the nature of the solvent, affect the way in which the material behaves in that solvent. Without wishing to be bound by theory, the panicles generally increase in size until they reach a colloidally stable state during the nanoprecipitation process.

As exemplified below, the present invention allows the encapsulation and release of not only organic materials ···· e.g. nile red, simulating encapsulation of a drug - but also inorganic materials - e.g. magnetic particles. This expands the utility of the present invention to cover further therapeutic and targeting uses. The encapsulation of inorganic material (e.g. magnetic material, e.g. iron oxide) in poiydendrons may also be considered as a standalone invention within mis disclosure.

The branches are typically distributed statistically throughout the connected linear polymer chains (rather than discretely in block polymerised monofunctional vinyl monomers and difunctional vinyl monomers). Each branch may be a glycol diester branch, for example.

The difunctional vinyl monomer acts as a brancher (or branching agent) and provides a branch between adjacent polymer chains. The branching agent may have two or more vinyl groups.

The monofunctional monomer utilised for the primary chain may comprise any carbon-carbon unsaturated compound which can be polymerised by an addition polymerisation mechanism, for example vinyl and ally! compounds . The

monofunctional monomer may be hydrophilic, hydrophobic, amphophilic, anionic, cationic, neutral or zwitteriomc in nature.

The monofunctional monomer may be selected from but is not necessarily limited to monomers such as: vinyl acids and derivatives (including esters, amides and anhydrides), vinyl aryl compounds, vinyl ethers, vinyl amines and derivatives (including aryl amines), vinyl nitrites, vinyl ketones, and derivatives of the aforementioned compounds as well as corresponding ally] variants thereof. Vinyl acids and derivatives thereof include: (metii)acrylic acid, fumaric acid, maleic acid, itaconic acid and acid halides thereof such as (meth)acryloyl chloride.

Vinyl acid esters and derivatives thereof include: CI to C20 alkyl(meth)acrylates (linear and branched) such as for example methyl (meth)acr late, stearyl

(meth)acrylate and 2-ethyl hexy! (meth)acrylate; aryl(meth)acrylates such as for example benzyl (meth)acrylate; tri(aikyioxy)silylalkyl(meth)acrylates such as rrimethoxysilylpropyl(meth)acryiate; and activated esters of (meth)acrylic acid such as N-hydroxysuccinamido (meth)acrylate. Vinyl aryl compounds and derivatives thereof include: stymie, acetoxystyrene, styrene sulfonic acid, 2- and 4- vinyl pyridine, vinyl naphthalene, vinylbenzyl chloride and vinyl benzoic acid.

Vinyl acid anhydrides and derivatives thereof include: nialeie anhydride.

Vinyl amides and derivatives thereof include: (meth)acrylamide, N-(2- hydroxypropyi)meihacrylamide, N-vinyl pyrrolidone, N-vinyi formamide, (meth)acrylamidopropyl triraethyl ammonium chloride, [3- ((me )acrylamido)propyl]dimethyl ammonium chloride, 3-[ -(3- (meth)acrylamidopropyi)-N,N-dimethyl]aininopropane sulfonate, methyl

(meth)acrylamidoglycolate methyl ether and N-isopropyl(meth)acrylamide.

Vinyl ethers and derivatives thereof include: methyl vinyl ether.

Vinyl amines and derivatives thereof include: dimethylaminoethy! (raeih)acrylaie, diethylammoethyl (mefh)aerylate, diisopropylaminoethyl (meth)acrylate, mono-t- hiitylaminoethyl (meth)acrylate, morpholinoethyl(meth)acrylate and monomers which can be post-reacted to form amine groups, such as N-vinyl formamide. aryl amines and derivatives thereof include; vinyl aniline, 2 and 4- vinyl .ne, N-vinyl carbazole and vinvl imidazole.

Vinyl nitriles and derivatives thereof include: (meth)acryionitrile. Vinyl ketones or aldehydes and derivatives thereof include: aereo

Monomers based on styrene or those containing an aromatic functionality such as styrene, a-methyl styrene, vinyl benzyl chloride, vinyl naphthalene, vinyl benzoic acid, N-vinyl carbazole, 2-, 3- or 4- vinyl pyridine, vinyl aniline, acetoxy styrene, styrene sulfonic acid, vinyl imidazole or derivatives thereof may also he used. Other suitable monofunctional monomers include; hydroxyl-containing monomers and monomers which can be post-reacted to form hydroxy! groups, acid-containing or acid-functional monomers, zwitterionic monomers and quateraised amino monomers.

Hydroxyl-containing monomers include: vinyl hydroxy! monomers such as hydroxyethyl (meth)acrylate, 1^■· and 2-hydroxy propyl (ntet.h)acrylate, 2-hydroxy raethacrylamide, glycerol raono(meth)acrylate and sugar mono(meth)acryiates such as glucose mono(meth)acrylate,

Monomers which can be post-reacted to form hydroxy! groups include: vinyl acetate, acetoxystyrene and glycidyl (meth)acrylate.

Acid-containing or acid functional monomers include: (meth)acrylic acid, styrene sulfonic acid, vinyl phosphonic acid, vinyl benzoic acid, maleic acid, fumaric acid, itaconic acid, 2-(metli)acrylamido 2-ethyl propanesulfonic acid, mono-2- ((meth)acryloyloxy)ethy{ succinate and ammonium sidfatoeiliyl (meth)acryiate.

Zwitterionic monomers include: (meth)acryloyl oxyethylphosphoryi choline and beiaines, such as [2-((meth)aeryloyloxy)ethyl] dimethyt-(3-sulfopropyl)ammomum hydroxide.

Quatemised amino monomers include: (meth)acryloyloxyethyltri- (alk aryl)ammonium haiides such as (meth)acryloyloxyethyitrimethyl ammonium chloride.

Oligomeric, polymeric and di- or multi-functionalised monomers may also be used, especially oligomeric or polymeric (meth)acrylic acid esters such as mono(a!k/aryl) (mem)aery!ie acid esters of polyalkyleneglycol or polydimethylsiloxane or any other mono-vinyl or al!yl adduct of a low molecular weight oligomer.

Oligomeric and polymeric monomers include: oligomeric and polymeric

(meth)acrylic acid esters such as mono(alk aryl)oxypolyalkylenegiycol (meth)acrylates and mono(aik'^'aryl)oxypolydimei l-siloxane(meih)acrylates. These esters include for example: monomethoxy oligo(ethyleneglycol)

mono(meth)acrylaie, monomethoxy oligo(propylenegl ycol) mono(meth)acrylate, monohydroxy oligo(ethyleneglycol) mono(meth)acrylate, monohydroxy

oligo(propyleneglycol) mono(meth)acrylate, monomethoxy poly(ethyleneg].ycol) mono(meth)acrylate, monomethoxy poly(propyleneg!yeoi) mono(meth)acrylate, monohydroxy poiy(ethyleneglycol) mono(meth)acrylate and monohydroxy poly(propyleneg!yeol) mono(meth)acr>'laie. Vinyl acetate and derivatives thereof can also be utilised.

Further examples include: vinyl or a!l l esters, amides or ethers of pre-formed oligomers or polymers formed via ring-opening polymerisation such as

oHgo(capro!aciam), oligo(eaproiaetone), poly(caprolactam) or po!y(eaprolactone), or oligomers or polymers formed via a living polymerisation technique such as polyi 1 ,4-butadiene).

The corresponding ally! monomers to those listed above can also be used where appropriate,

Specific examples of monofunetiona! monomers include: amide-containing monomers such as (meth)acryl amide, N-(2-hydroxypropyl) raethacrylamide, N and/or N'-di(alkyl or aryl) (metli)acryl amide, N-vinyl pyrrolidone, [3-((meth)acrylamido)propyl] trimethyl ammonium chloride, 3-(dimethylamino)propyl(meth)acrylamide, 3~ N-(3- (meth)aerylaniidopropyl)~N,N-diine hyi]aminopropane sulfonate, methyl

(meth)acrylamidogiyco!ate methyl ether and N-isopropyl(meth)acrylamide; (meth)acrylic acid and derivatives thereof such as (meth)acrylic acid, (metli)acryloyl chloride (or any halide), (aikyl/aryl)(meth)acrylate; vinyl amines such as aniinoethyl (meth)acrylate, dimethyl aminoethyl (meth)aeryiate, diethylaminoethy! (meth)aery!ate, dusopropylaminoethyl (meth)acrykte, mono-t- butyl amino (meth)acrylate, morpholinoeihyl(rneih)aeryIafe; vinyl aryl amines such as vinyl aniline, vinyl pyridine, N- vinyl carbazoie, vinyl imidazole, and monomers which can be post-reacted to form amine groups, such as vinyl formamide; vinyl aryl monomers such as styrene, vinyl benzyl chloride, vinyl toluene, alpha- methyl styrene, styrene sulfonic acid, vinyl naphthalene and vinyl benzoic acid;

10 vinyl hydroxy! monomers such as hydro yethyl (meth)acrylate, hydroxy propyl (meth)aeryiate, glycerol mono(meth)acrylate or monomers which can he post- functionalised into hydroxy! groups such as vinyl acetate, acetoxy styrene and gl cidyl (meth)acrylate;

I S acid-containing monomers such as (meth)acrylic acid, styrene sulfonic acid, vinyl phosphonic acid, vinyl benzoic acid, maleie acid, fumaric acid, itaconic acid, 2- (meih)acrylamido 2-ethyl propanesulfonic acid and mono-2- ((meth)acryloyioxy)ethyl succinate or acid anhydrides such as maleic anhydride;

20 zwitterionic monomers such as (meth)acryloyl oxyethylphosphoryl choline and betaine-conraining monomers, such as [2-((meth )acryloyloxy)ethyi] dimei^'hyl-(3~ sulfopropyl)ammonium hydroxide; quateroised amino monomers such as (meth)acryioyloxyethyltrimethyl ammonium 25 chloride, vinyl acetate or vinyl butanoate or derivatives thereof.

The corresponding ally! monomer, where applicable, can also be used in each case.

Mixtures of more than one monomer may also be used to give statistical, graft, gradient or alternating copolymers. Some preferred monofunctional vinyl monomers include methacrylate monomers or styrene. Some preferred hydrophobic methacrylate monomers include 2- hydroxypropyl methacrylate (ΗΡΜΆ), n-butyl methacrylate (nBuMA), tert-butyl methacrylate (tBuMA), and oligo(ethy!ene glycol) methyl ether methacrylate (OEGMA). HP MA is particularly preferred, and is readily available or synthesised as a mixture of (predominantly) 2~hydroxypropyl methacrylate and 2- hydroxyisopropyl methacrylate, A preferred hydrophilic methacrylate monomers is diethylaminoeihyl methacrylate (DEAEMA). The polydendron also contains a brancher which is a multifunctional (at least difunctional) vinyl containing molecule.

The multifunctional monomer or brancher may comprise a molecule containing at least two vinyl groups which may be polymerised via addition polymerisation. The molecule may be hydrophilic, hydrophobic, amphophilic, neutral, eationie, zwitterionie, oligomeric or polymeric. Such molecules are often known as cross- linking agents in the art.

Examples include: di- or multi vinyl esters, di- or mu ti vinyl amides, di- or multivinyl ary compounds, di- or multivinyl alk aryl ethers. Typically, in the case of oligomeric or polymeric di™ or multifunctional branching agents, a linking reaction is used to attach a poiymerisable moiety to a di- or multifunctional oligome or polymer. The brancher may itself have more than one branching point, such as T- shaped divinylic oligomers or polymers. In some cases, more than one

multifunctional monomer may be used. The corresponding ally! monomers to those listed above can also be used where appropriate.

Preferred multifunctional monomers or branchers include hut are not limited to: divinyl aryl monomers such as diviny! benzene; (meth)acrylate diesters such as ethylene glycol di(meth)acrylale, propyleneglycol di(meth)acrylate and 1 ,3- hutylenedi(meth)aerylate; poiyaikylene oxide di(meth)acrylates such as tetraethyleneglycol di(meth)acry1ate, poly(ethyleneglycol) di(meth)acrylate and poly(propyleneglycol) di(meth)acrylate; divinyl (raeth)acrylamides such as methylene bisacryl amide; silicone-contaraing divinyl esters or amides such as (meth)acryloxypropyl -terminated

poly(dimethylsiloxane); divinyl ethers such as poly(ethyleneglycol)di vinyl ether; and terra- or iri-(meth)acrylate esters such as pentaerythritol tetra(meth)acrylate, triraethylolpropane tri(raeth)acrylate or glucose di- to penta(meth)acrylate.

Further examples include: vinyl or ally! esters, amides or ethers of pre-formed oligomers or polymers formed via ring-opening polymerisation such as

oligo(caprolactarn), oligo(caproiactone), poly(eaprolaelam) or poly(caprolactone), or oligomers or polymers formed via a living polymerisation technique such as oligo- or poly(] ,4-butadiene).Some preferred types of difunctional vinyl monomers include dimethacrylate monomers, for example ethyleneglycol diraethacrylate (EGDMA).

The molar ratio of difonctional vinyl monomer to initiator is preferably no more than 2, more preferably no more than 1.5, and most preferably no more than 1 if conducted under appropriate conditions. The amount of difunctional vinyl monomer relative to monofunctionai vinyl monomer is preferably 7.5 mol% or less, 2 mol% or less, or 1.6mol% or less, more preferably between 1 and 7.5 mol%, for example between 1 and 2 mol %

In a preferred embodiment, the method is a one-pot method, In this embodiment, the reaction of monofunctionai vinyl monomer, difunctional vinyl monomer and initiators is carried out conveniently and cost-effectively.

Preferably the method comprises preparing a mixture of the monofunctionai vinyl monomer, difunctional vinyl monomer and initiators under suitable conditions. The mixture may contain a catalyst (such as CuCI) or additional agents depending on the addition polymerisation technique being used. The mixture may also contain a ligand (such as 2,2'-bip ridine). The mixture may also contain a chain transfer agent. Suitable ATRP initiators include isobutyrate esters, preferably haloisobutyraie esters, most preferably hromoisobutyrate esters. Thus the initiator can for example have the following genera] formula Ϊ:

I wherein X denotes a chemically addressable group and is preferably a halide, for example CI or Br, most preferably Br; and wherein is any suitable organic moiety. Where the initiator is a dendron initiator, R. is branched into a dendritic wedge and X is the chemically addressable group at the apex of the dendritic wedge. Whilst isohutyryi esters are convenient and effective to use in this context, other chemistries are possible.

It of course will be understood that part of the initiator (in this ease the X group, usually bromide) is present in the initiator but reacts during the process so that it is not necessarily present in the product at the end of all primary chains.

Where the initiator of general formula I is a dendron initiator, R is a moiety which divides into two or more (preferably two) first generation branches (preferably identical first generation branches). Optionally each of those first generation branches then divides into two or more (preferably two) second generation branches (preferably identical second generation branches). Optionally each of those second generation branches then divides into two or more (preferably two) third generation branches (preferably identical third generation branches). There may analogously be further generations of branching, A dendron having only first generation branches is known as a generation 1 dendron; a Dendron having first and second generation branches is known as a generation 2 dendron.

The outermost branches of the dendron (the part most likely to end up on the surface of the polydendron) may comprise one or more of a variety of chemical groups, for example aromatic groups (e.g. benzene rings, e.g. of benzyloxy groups), amines (e.g. tertiary amines), aikyl groups (e.g. aikyi chains or branched a!kyl groups e.g. tertiary butyl groups), amide groups, xanthates or carbamates (e.g.terminatiag in a tertiary butyl group). These are however merely non-limiting examples: many chemistries are possible. One of the advantages of the present invention is that is compatible with a wide variety of different types of dendrons and other groups; ihe flexibility provided by the use of mixed initiators is considerable. The properties can be tuned by selecting dendrons with different chemical constituents and/or different surface groups, for example hydrophilic or hydrophobic groups, large or small moieties, groups of different polar or electronic character, groups which may allow further conjugation, etc..

Each, segment may comprise one or more of an alky! chain, ester, carbamate, or other linking group. Again these are merely non-limiting examples and many chemistries are possible,

Within the dendron, the structure may divide at any suitable point, for example a carbon atom or a nitrogen atom, or a larger moiety such as a ring. For example the structure may comprise a N₅N~bis~substituted amino component, e.g. esters of 1- [Ν,Ν-bis-substituted amino] -2-propanol.

Some specific and non-limiting examples of possible dendrons will now be described.

A first ciass of possible dendrons include those having benzyloxy surface groups. For example the surface tructure:

Optionally two of these moieties may be linked via carbamate chains to an amide branching point. Examples in ibis class of dendrons include the Gl and G2 structures shown in Figure

A second class of possible dendrons include those having tertiary amine surface groups, for example where the end amines are dimethyl substituted. Optionally the branching may occur at tertiary amine centres and the segments may contain ester linkages.

Examples in this class of dendrons. and a suitable component thereof, are shown in Figure 2.

A third class of possible dendrons include those having carbamate surface functionality, for example tertiary butyl carbamates, and optionally carbamate functionality within the segment(s).

Examples in this class of dendrons are shown in Figure 3,

A fourth class of possible dendrons include those having xanthate functionality, optionally with branches comprising esters.

Examples in this class of dendrons, and a suitable component thereof, are shown in Figure 4.

The dendrons may be prepared by known chemical techniques. Some possible methods of preparation include those described below.

The present invention will now be described in further non-limiting detail and with reference to the Examples and Figures in which: Figures 1 to 4 show some examples of dendron initiators and components thereof used in the present invention; Figure 5 shows, schematically, structural differences between dendrimers and polydendrons;

Figure 6 and 7 show MTT assays of Caco-2 cells following incubation with aqueous Nile Red and polydendrons;

Figure 8 and 9 show ATP assays of Caco-2 cells following incubation with aqueous Nile Red and polydendrons;

Figure 10 shows results in relation to transcellular permeability of selected Nile Red polydendron materials across Caco-2 cell monolayers

Figure 1 1 shows, schematically, how using different dendron : polyethylene giycoi initiator ratios can result in a spectrum of hydrophobicity, ampbipbilicity and liydrophiiicity;

Figure 12 is a photograph, corresponding to Figure 1 1 , and illustrates how using different dendron : polyethylene glycol initiator ratios can affect the response of encapsulated Nile Red;

Figure 13 shows, schematically, one method of nanoprecipitation of polydendrons;

Figures 14a and 14b are SEM images of polydendron nanoprecipitates.

The experimental details below relate to: preparative procedures for various dendron and non-dendron initiators used in the present invention, including initiators containing polyethylene glycol (PEG) and sugar moieties; preparative procedures and properties of various polydendrons showing how hydrophilic or hydrophobic properties can be tailored; nanoprecipitation methods and results; encapsulation experiments showing how molecules can be encapsulated and showing the effect of tailoring the encapsulation environment, as a model for drug encapsulation;

cytotoxicity analysis using MIT and ATP assays in respect of Caco-2 cells;

transcellular permeability of polydendrons carrying Nile Red (to model drug transfer across the intestinal epithelium); and encapsulation of inorganic material (e.g.

magnetic particles).

Very positive results were obtained with regard to cytotoxicity and in the drug transport model. The experiments below show in particular that a material which would otherwise not pass effectively from gut to blood can be carried over by using polydendrons of the present invention.

Whereas a representation of an ideal dendrimer structure is shown in Figure 5a, the present invention is concerned with polydendrons which have dendrons and a polymer core as represented in Figure 5c, constituent parts of which include dendrons attached to polymer chains as represented in Figure 5b. The poiydendron represented in Figure 5c has several dendrons of the same type; however the focus of the present invention is on polydendrons which have a branched polymer core and which carry not only one type of dendron moiety but also at least one further moiety, whether that be a dendron moiety or a non-dendron moiety.

In other words the polydendrons can be prepared by using mixed initiators, to end up with poiydendron structures as represented for example in Figure 11. At the far left of Figure 11 is represented a hydrophilic poiydendron made using 100% dendron initiator; at the far right of Figure 1 1 is represented a hydrophobic material made using 100% PEG. The hydrophobicity/ amphiphilicity/ hydrophilicity can be tuned by varying the relative amounts of the different intiators.

Figure 12 is a photograph of vials containing the seven different types of

poiydendron shown schematically in Figure 11 (i.e. 100% dendron initiator with 0% PEG initiator on the left, through to 0% dendron initiator with 100% PEG initiator on the right) carrying Nile Red. In the original photograph, the darkest pink colour can be seen on the left, lighter pinks in the middle vials, and a very pale pink on the right, thereby showing that the hydrophobicity can be tuned in a discernible and

control! able m anner. Whilst the present invention is primarily focused on the use of mixed initiators to prepare poiydendrons, and the products themselves, nevertheless the present invention also covers the corresponding methods and products wherein only one type of initiator is used, in other words where one of the dendron initiators disclosed herein is used.

Novel products, components thereof, intermediates, methods or method steps, disclosed herein, also fall within the scope of the present invention Y AMPT P¾ sv hi

Lactose (4 g, 1 1.7 mmol) was weighed into a 100 mL round bottom lla.sk equipped with a magnetic stirrer and dry N₂ inlet. The flask was purged with nitrogen for 15 minutes. Acetic anhydride (30 mL) and odine (208 nig, 1.58 mmol) were added, instantly forming a brown coloured solution. Within 10 minutes the flask began to warm due to onset of acetylation. The solution was stirred overnight at room temperature under a positive flow of nitrogen. The solution was transferred to a 250 nil, separating funnel containing dicbiorome hane (50 mL), sodium thiosulfate solution (30 mL) and crushed ice, and the product was extracted into the organic layer. The aqueous layer was further extracted with dich!oromethane (2x50 mL). The organic phases were collected and washed with saturated sodium carbonate solution until neutral. The organic phase was collected, dried over anhydrous MgSOi, and concentrated in vacuo to give a white solid. Ethylene Dsass¾sse

Acetic Acid, THF

Lactose octa-acetate (5.1 g, 7.52 mmoi) was weighed into a 250 mL round bottom flask equipped with a magnetic stirrer, and was dissolved in tetrahydrofuran (100 mL). Ethylene diamine (0.6 mL, 9.02 mraol) was added to the flask, followed by the slow addition of acetic acid (0,6 mL, 10,5 mrool), to give a white coloured turbid solution, A gas was evolved and the flask wanned slightly upon addition of the acid. The flask was lightly sealed with a rubber septum cap, and stirred overnight at room temperature, to give a cream coloured mixture. Distilled water (50 mL) was added to the flask, whereby the precipitate dissolved, leaving a slightly yellow coloured solution. The solution was transferred to a 500 mL separating funnel contai ing dichloromethane (100 mL), and the product was extracted into the organic solvent. A further extraction of the aqueous layer was performed with dichloromethane (50 mL). The organic layers were combined, washed with hydrochloric acid (80 mL, 2M), saturated sodium bicarbonate solution (80 mL) and distilled water (80 mL). 'The organic layer was dried over anhydrous MgS<¾, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (silica, eluent hexane/acetone, 60/40) to give a white solid.

Lactose septa-acetate (3 g, 4.71 mmol) was added to a 50mL round bottom flask equipped with a magnetic stirrer and dry N? inlet. The flask was then purged with nitrogen for 10 minutes. Anhydrous tetrahydrofuran (8 mL) was added to the flask, and 2 was bubbled through the mixture for a further 10 minutes, Trieihyla nine (0.99 mL, 7.07 mmoi) was added to a vial, diluted with tetrahydrofuran (2 mL), and then transferred to the reaction flask drop-wise. Following this, 2-bromoisobutyryl bromide (0.87 mL, 7.07 mmol) was added to a vial, diluted with tetrahydrofuran (2 ml..) and transferred to the reaction flask drop- wise. Reaction mixture was left to stir overnight at room temperature under a positive flow of nitrogen. This gave a white coloured turbid mixture. The mixture was filtered by gravity filtration, the precipitate washed with tetrahydrofuran, and the solution concentrated in vacuo. The crude product was purified by flash column chromatography (silica, eluent hexane/ethyl acetate, 95/5) to give a white solid,

L2 PEG initiators

1.2, 1 750-PEG initiator

Monomethoxy polyethylene glycol) (Mw ~ 750 gmol^" ) (23.0 g, 30.7 romol) was dissolved in warm THF (-40 °C), and the reaction was degassed with dry Na. DMAP (37.5 mg, 0.3 mmol) aid TEA (7.48 ml. 53.7 mmol) were added and the reactio was cooled to 0 °C in an ice bath, a-bromo isobut ryi bromide (5,69 ml, 46.0 mmol) was added dropwise over 30 minutes and a white precipitate appeared immediately; the Ι¾Ν1 Β salt. After 24 hours the precipitate was filtered, THF removed in vacuo and the resulting crude product was precipitated from acetone into petroleum ether (30-40 °C) twice (72 %), Ή NMR (400 MHz, D₂0) δ ppm 4,31 (m, 2H), 3.77 (m, 2H), 3.70-3.59 (m, 60H), 3,55 (m, 2H), 3.31 (s, 3H) and 1.89 (s, 6H).

1.2,2 2 -PEG initiator

. RT 24hrs

Monomethoxy poiy( ethylene glycol) (Mw ~ 2000 gmoi^"1) (20,5 g. 10.25 mmoi) was dissolved in warm THF (-40 °C), and the reaction was degassed with dry N-->. DMAP (12.5 mg, 0.1 mmoi) and TEA (3.14 ml, 22.5 mmol) were added and the reaction was cooled to 0 °C in an ice bath. a~bromo isobuiyryl bromide (2.53 ml, 20.5 mmol) was added dropwise over 20 minutes and a white precipitate appeared immediately; the Et₃NH⁺Br^" salt. After 24 hours the precipitate was filtered, THF^' removed in vacuo and the resulting crude product was precipitated from acetone into petroleum ether (30-40 °C) twice (89 %). Ή MR (400 MHz, D₂0) δ ppm 4.34 (m, 2H)₅ 3.80-3.59 (m₅ 186H), 3.35 (s, 3H) and 1.93 (s, 6H).

1.3.1 GO Tertiary amine functional initiator

o l-dimethylamino-2-propanoi (1 ,1207 g, 10,86 mmol, 1 eq.), TEA (1 ,5390 g, 15.2 mmol 1.4 eq.) and DMAP (i 32.7 mg, 1.086 mmol, 0.1 eq.) were added to a 250 ml, 2 necked round-bottomed flask containing DCM (160 mL). The flask was deoxygenated under a positive N2 purge tor 10 minutes, a-bromoisobutyryl bromide (2.622 g, 1.4 mL, 1 1.4 mmol, 1.05 eq.) was added drop wise while the solution was stirring in an ice bath under a positive flo of . The reaction mixture was allowed to warm to room temperature and left stirring overnight. The organic phase was washed with saturated sodium hydrogen carbonate ( aHCOs) solution (3 x 30 mL). The solution was dried with anhydrous Na₂S0 . Ή NMR (400 MHz, CDC¾) δ 1.27 (d, 3H), 1.89 (m, 6H), 2.17-2.55 (m, 8H), 5.07 (m, 1H). m/z (ES MS) 252 [M+H^" .

1 ,3~Dibenzyioxy-2-propanol, 1, (9.80 g, 36.0 mmol) was weighed into a 2-neck round bottom flask which was equipped with magnetic stirrer and dry N₂ inlet.

Dichlororneihane (DCM) (100 ml) was added followed by 4- (dimethylamino)pyridine (DMA?) (0.44 g, 3.6 mmol) and triethylamine (TEA) (7.53 ml, 54.0 mmol). The reaction was cooled to 0 °C in an ice-bath and a- bromoisobutyryl bromide (534 ml, 43.2 mrnoi) was added dropwise over 20 minutes. Alter complete addition the reaction was warmed to room temperature and left stirring overnight. Reaction could be observed by the formation of a white precipitate. After 2.4 hours the precipitate was removed by filtration, the resulting crude reaction medium was washed first with a saturated solution of NaHC(¾ (3 x 100 ml) followed by distilled water (3 x 100 ml). The organic layer was dried over ¾3θ4 and concentrated in vacuo to give a pale yellow oil (81 %). Found, C, 59.55; H, 6.02 %. C_2JH₂₅Br0₄ requires, C, 59.86; H, 5.98; Br, 18.96; O, 15.19 %. Ή NMR (400 MHz, CDCI₃) δ ppm 7.35-7.20 (m, 10H), 5.26 (ni 1 H), 4.55 (m, 4H), 3.69 (d, 4H), 1.93 (s, 6H). ^! C NMR (100 MHz, CDC1₃) δ ppm 171.2, 138.0, 128.4, 127.7, 127.6, 73.3, 68.5, 55.8, 30.7. m/z (ES MS) 443.1 [M+Na , 461.1 [M+KJ\ m/z required 420,1 [MJ \ .4.2 G2~ aromatic dendron ini l

1 ,1 ' -Carbonyldiimidazoie (GDI) (9.73 g, 60.0 mmol) was weighed info a 2 -neck round bottom flask and equipped with magnetic stirring, condenser and dry inlet. Anhydrous toluene (100 ml) was added, followed by KOH (0.34 g, 6,0 mmol) and 1 (12.35 ml, 50.0 mmol). The reaction was heated to 60 °C for 6 hours. Toluene was removed in vacuo, the crude mixture was dissolved in DCM (50 ml) and washed with distilled water (3 x 50 ml). The organic layer was dried over N -jSil* and concentrated in vacuo to give 3, a pale yellow oil (97 %). Found C, 68.64; H, 6.10; N, 7.85 %. C21H22N2O4 requires C, 68.84; H, 6.05; N, 7.65; O, 7.47 %. Ή NMR (400 MHz, CDCI₃) δ ppm 8.1 1 (s, I H), 7.41 (s, I H), 7.33-7.23 (m, 10H), 7.06 (s, IH), 5.36 (qn, IB), 4.53 ( , 4H), 3.75 (m, 4H). ^!3C NMR ( 100 MHz, CDC¾) δ ppm 148.3, 137.5, 137.2, 130.6, 128.4, 127.9, 127.6, 1 17.2, 76.1 , 73.3, 68.1. /z (ES MS) 367.2 [M-i-H , 389.2 405.1 [M+ , m/z required 366.2 [M]⁺. 3 (16.84 g, 46.0 mmol) was weighed into a 2. -neck round bottom flask which was equipped with magnetic stirring, condenser and dry Ν·? inlet. Anhydrous toluene (120 mi) was added followed by diethyienetriamine (DETA) (2.48 ml, 23.0 mmol). The reaction was heated to 60 °C for 48 hours. Toluene was removed in vacuo, the resulting crude mixture was dissolved in DCM (100 ml) and washed with distilled water (3 x 100 ml). The organic layer was dried over and concentrated in vacuo to give 4, a yellow oil (93 %). Found C, 68.50: H, 7.13; N, 6.00 %.

C40H49 3OS requires, C, 68.65; H, 7.06; N, 6.00; 0, 18.29 %. Ή NMR (400 MHz, CDCI₃) δ ppm 7.27-7.16 (m, 20H), 5.23 (s, br, NH), 5.03 (qn, 2H), 4.44 (m, 8H), 3.57 (d, 8H), 3.12 (m, 4H), 2.58 (m_} 4H). "C NMR. (100 MHz, CDC1₃) 5 ppm 156.6, 138.4, 128.8, 128.1, 73.7, 72.1, 69.4, 49.0, 41.2. m/z (ES MS) 700.4 [M+Hf, 722.3 [M+ a]⁺, 738.3 [M+K]⁺, m/z required 699.4 [M]\

4 (15.01 g, 21.4 rnmol) was weiglied into a 2 -neck round bottom flask, equipped with magnetic stirrer, condenser and dry N₂ inlet. Anhydrous toluene (90 ml) was added followed by dropwise addition of β-butyrolactone (2.62 ml, 32.2 mmol). The reaction was heated at reflux for 16 hours. Toluene was removed in vacuo, the resulting crude mixture was dissolved in DCM (50 ml) and washed with distilled water (3 x 50 ml). The organic layer was dried over Na.2S04 and concentrated in vacuo to give a yellow oil. The crude product was purified by silica gel column chromatography with a mobile phase gradient of DCMrMeOH (100:0 - 95:5 - 90:10) to give 5. a pale yellow oil (45 %). Found C, 65.35; H, 6.72; N, 5.10 %. C44H55N3O1Q requires, C, 67.24; H, 7.05; N, 5.35; O, 20.36 %. Ή NMR (400 MHz, CDC¾) δ ppm 7.34-7.25 (m, 20H), 5.35 (br, NH), 5.31 (br, NH), 5.1 1 (m, 2H), 4.50 (m, 8H), 4.14 (s, 1H), 3.62 (m, 8H), 3.46-3.18 (m, br, 8H), 2.45-2.22 (m, 2H), 1.18-1.05 (m, 3H). ^tsC NMR. (100 MHz, CDC1₃) δ ppm 174.4, 156.8, 1.56.6, 138.4, 138.3, 128.8, 128.1, 128.0, 73.7, 73.6, 72.6, 72.4, 69.5, 69.3, 65.1 , 48.5, 46.5, 41.2, 40.3, 39.9, 22.9. m/z (ES MS) 808.4 iWNa) \ m/z required 785.4 [M]⁺. 5 (9.31 g, 1 1.85 mmo!) was dissolved in DCM (100 ml) and transferred to a round bottom flask which was equipped with magnetic stirring and a dry N_∑ inlet. DMAP

(0.1.4 g, 1.19 mmol), TEA (3.30 m!, 23.7 mmol) were added and the reaction mixture was cooled to 0 °C in an ice bath followed by dropwise addition of a- bromoisobutyryl bromide (2.19 ml, 17.78 mmol). The reaction was warmed to room temperature for 24 hours. A colour change from pale orange to a dark orange/brown colour was observed over time. No precipitate was observed, the cmde reaction mixture was washed with a saturated NaHCOs solution (3 x 100 nil) and distilled water (3 100 ml). The organic layer was dried over and concentrated in vacuo to give 6, an orange oil (81 %). Found C, 59.50; H. 6.31 ; N, 4.39 %.

C₄sH₆oBrN₃O_u requires, C, 61.67; H, 6.47; Br, 8.55; N, 4.49; O, 18.82 %. Ή NMR (400 MHz, CDC1₃) δ ppm 7,35-7.23 (m, 2011), 5.33 (s, br, NH), 5.10 (m, 2H)_; 4.52 (m, 8H), 3.71-3.53 (s_s 8H), 3.52-3.12 (m, br, 8H), 2.76 (d of d, 1H), 2.47 (d of d, 1 H), 1.87 (s, 6H), 1.29 (d, 3H). ^iJC NM (100 MHz, CDC¾) δ ppm 192,5, 170,8, 156.3, 156.1 , 137.9, 134.5, 128.4, 127.7, 127.6, 73.2, 73.1 , 72.2, 71.8, 70.2, 69.1, 69.0, 68.8, 56.1, 48.3, 46.3, 39.6, 39.4, 38.9, 30.8, 30.7, 30.6, 1 .7. mh (ES MS) 958.3 [M-^;-Na]⁺, 974.3 [M+K]\ mJz required 933.3 [M^' .

3 (14.03 g, 38.3 mmol) was added to a 2-neck round bottom flask, which was equipped with magnetic stirring, condenser and a N? inlet. Anhydrous toluene (1 0 ml) was added and the reaction was heated to 60 °C. The AB? brancher (3.627 g, 19.2 mmol) was dissolved in anhydrous toluene (5 ml) was added dropwise. After 18 hours the reaction was stopped, the toluene removed in vacuo, the crude mixture was dissolved in diehloromethane (100 ml) and washed with water (3 x 100 ml). The organic phase was dried over Na-?80 the solvent removed in vacuo and the resulting yellow oil was dried further under high vacuum to give 7, as a pais yellow oil, (94 ). Ή NMR (400 MHz, CDC1₃) δ ppm 7.33-7.23 (m, 20H), 5.30 (s, br, NH), 5.09 (m, 2H)₃ 4.51 (m, 8H), 3.73 (m, 1H), 3.64 (d, 8H), 3.16 (m, 4H), 2.53 (m, 2H), 2.32 (m, 2H), 2.24 (m, 2H), 1.59 (m, 4H), 1.(56 (d, 3B). m/z (ES MS) 786.4

808.4 [ ' a]⁺, m/z required 785.43 [Mf.

7, (13.381 g, 17.0 mmol) was dissolved in DCM (100 ml) and bubbled with N₂ for 20 minutes. 4-(Dimethylamino)pyridine (DMAP) (21 mg, 0,17 mmol) and triethylamine (TEA) (3,56 ml, 26.0 mmol) were added and the reaction vessel was cooled to 0 °C. a-Bromoisobutyryi bromide (2.53 mi, 20.0 mmol) was added, dropwise, then the reaction was warmed to room temperature for 24 hours. The organic phase was washed with a saturated solution of NaH€i¾ (3 x 150 ml) and distilled water (3 x 150 mi), dried over N iSCXt and the solvent removed in vacuo to give an orange oil as the crude product. This was purified by column

chromatography with a silica stationary phase and mobile phase of ethyl

acetaterhexane (4: 1), to give 8 a yellow oil, (73 %). Found C, 63,24: Pi, 6.88; N, 4.44 %. C ₉H₆ BrN₃Oio requires, C, 62.95; H, 6.90; N, 4.49 %. Ή NMR (400 MHz, CDC ) δ ppm 7.33-7,24 (m, 20H), 5.36 (s, br, NH), 5,09 (m, 2H), 5.03 (ra, I B), 4.51 (m, SH), 3.64 (d, 8H), 3.16 (m, 4H), 2.64-2.35 (m, 6H), 1.89 (s, 6H), 1 .60 (m, 4H), 1.22 ( 3H). »C NMR (100 MHz, CDC1₃) δ ppm 171.2, 156.0, 138.1, 12.8.3, 127.60, 127.62, 73.2, 71.6, 70.4, 68.9, 59.1, 56.1, 52.2, 39.4, 30.6, 30.7, 27.2, 18.0. m/z (ES MS) 936.4 [M+H]\ 959.4 fM+Na]*, m/z required 935.4 [M]⁺,

5 Ae e 4 --- Synthesis of 20 (2~(B (3~aminopropyl)aniino)propan--! -ol) using CDI chemistry Synthesis of 18 - CDI (39.137g, 0,241 mol) was added to an oven-dried 50QmL 2- neck RBF fitted with a reflux condenser, magnetic stirrer and a dry Nz inlet. Dry toluene (350mL) was added and the flask was purged with N? for 10 minutes. The solution was stirred at 60°C and 17 (t-Butanol) (35.7g, 46mL, 0.483 mol) was added via a warm syringe. The mixture was left stirring at 60°C for 6 hours under a positive flow of nitrogen. Following this, BAPA ( 16.077g, 17.14mL, 0.121 mol) was added dropwise. The reaction was left stirring for a further 18 hours at 60°C under a positive flow of nitrogen, and then allowed to cool to room temperature. The pale yellow solution was filtered to remove any solid imidazole, and concentrated in vacuo. The remaining oil was dissolved in dichloromethane (250m.L) washed with distilled water (3 x 250mL) and finally a saturated brine solution (150mL). The organic layer was dried with anhydrous N ?SC)4, filtered and concentrated in vacuo to give 18 as a white solid powder. 38g, (95%) Found C, 57.84; H, 10.45; N, 12.91%. C16H33N3O4 requires, C, 57.98; H, 10.04; N, 12.68%. 1H NMR (400MHz, CDC1₃) 5.19 (s, br, NH ···· disappears on addition ofD₂0), 3.21 (t, 4H), 2.65 (t, 4H), 1.65 (q, 4H), 1 ,44 (s, 18H) ^l3C NMR (100MHz, CDCI3) 156.48, 79.34, 47.77, 39.29, 30.1 1 ,

28.79. m/z (ES MS) 332.3 [M+Hf Synthesis of 19 - 18 (20g, 0.06 mol) was added to a 500mL 2-neeked RBF fitted with a reflux condenser, magnetic stirrer and a dry N₂ inlet. The flask was degassed with dry nitrogen for 10 minutes, and dissolved in dry ethanol (2()()mL), Whilst stirring, and maintaining the temperature at 30°C, propylene oxide (10.51 g, 1 1.2^'lmL, 0.1 81 mol) was added dropwise over a period of 10 minutes. Under a positive flow of dry N₂, the reaction was left stirring at 30°C for 18 hours. After this time, the solvent and excess propylene oxide were removed in vacuo. The crude product was purified by liquid chromatography on silica gel, eluting with EtGAc:MeOH, 4:1, the solvent removed in vacuo to give 19 as a pale yellow viscous oil. 1 .90g, (85%) Found C, 58,50; H, 10.23; N, 10.82%. Q9H39N3O5 requires, C, 58.58; H, 10.09; N, 10.79%. Ή NMR (400MHz, CDCI₃) 4.93 (s, br, NH), 3,76 (m, I H), 3.15 (m, 4H), 2.61-2.88 (m, 6H), 1 ,62 (m, 4H), 1.44 (s, 18H), 1.1.1 (d, 3H).ⁿC NMR (100MHz, CDCI3) 156.08, 79.18, 63,45, 62.55, 51.77, 38.75, 27.48, 20.14. m/z (ES MS) 390.3 [M+Hf

Synthesis of 20 (Pari 1) - In a 1 L RBF, Gl-OH (33.70g) was dissolved in ethyl acetate (330mL) and concentrated HC1 (35.03g, 30mL, d^::::l .l 8 36% active) was added very slowly. CO₂ began to evolve. The reaction vessel was left open and stirring for 6 hours, ⁵H NMR (DjO) confirmed complete decarboxylation.

Synthesis of 20 (Part 2} ~ After removal of ethyl acetate, the crude oil was dissolved in 4M NaOH (300mL), and then reduced down by half (approx.) on the rotary evaporator {6Q°C). Following this, the oily mixture was extracted twice with CHC1₃ (300mL). The organic layers were then combined, dried with anhydrous N aSCU, filtered and concentrated in vacuo to give the product as a pale yellow oil (15.27g, 94% yield) NMR (400MHz, CDCI₃) 3.79 (m, 1 H), 2.68-2.40 (ddd, 2H), 2,31 (m, 4H), 1.89 (s, br, OH), 1 60 (m, 4H), 1.1.1 (d, 3H), C NMR (100MHz, CDC¾) 63.95, 62,56, 52.10, 40.31 , 30.80, 20,03

Preparation qft-BOC G2 dendron, 21

Scheme 5 Synthesis of 22, t-BOC G2 dendron

Synthesis of 21 - 19 (Sg, 12.8 rnmol.) was added to a 250mL 3 necked round bottom flask containing dry toluene (60mL), which was fitted with a reflux condenser, magnetic stirrer and a dry ½ inlet. The flask, was purged with hb for 10 minutes. The solution was stirred at room temperature and CD! (2.29g, 14.1 mmole) was added via a powder addition funnel. The mixture was heated to 60°C with stirring for 6 hours. 20 (0.9 IrnL, 6.4mmole) was added dropwise whilst the solution was stirring and the temperature was maintained at 60°C. The reaction was left overnight stirring for a further 12 hours at 60°C, and then allowed to cool to room temperature. The clear solution was tittered to remove any solid imidazole, and concentrated in vacuo. The crude product was purified by liquid chromatography, silica gel, eluting with EtOAc:MeOH, 5:1 , the solvent removed in vacuo to give 21 as a pale yellow viscous oii (60%) Found C, 57.46; H, 9.83; N, 12.17%. C_l9H_i9N 0₅ requires, C, 57.68; H, 9.58; N, 12.35%.^¾H NMR (400MHz, CDC1₃) 4.92 (m, br, 2H), 3.74 (ra, 1H), 3.35- 2.93 (m, 12H 2.73-2.14 (m, 18H), 1.62 (m, I 2H), 1.44 (s_s 36H), 1.20 (m, 6H), 1.10 (d, 3H) ⁱ³C NMR (100MHz, CDC1₃) 156.76, 156.15, 78.91 , 67.58, 63.51 , 62.46, 59.36, 52.33, 51.75, 38.94, 28,50, 27.37, 20.13, 18.82, 14.20. (ES MS) 1020.7 of t-BOC initiators 22 and 23

21

Scheme 5 - Synthesis of 22 and 23 t-BOC ATRP initiators

General Procedure for focal point modification to ATRP initiator by acid bromide - 19 or 20 was added to a 50mL round bottom flask, which was equipped with a magnetic stirrer and purged with dry N? for 10 minutes. Following this,

dichSoromethane (40m L), DMAP (0.2 eqv.) and TEA (2eqv.) were also added. The round bottom flask was then purged again with dry N?, and placed into an ice bath. Dropwise, over a period of 10 minutes 2-Bromoisobutyryl bromide (1 , 1 eqv.) was added. The reaction was removed from the ice hath after 30 minutes and left for 24 hours at room temperature. A colour change from clear to yellow/orange was noted for all reactions. After this time, the solution was filtered, washed with distilled water (3 x 4QmL), washed with a saturated brine solution (40mL) and the organic layer dried using anhydrous Ν¾8θ . The solvent was removed in vacuo, and the crude product purified by column chromatography

Synthesis of 22^■■■■ 19, Bromoisobutyryl bromide (1 , 1 eqv,), DMAP (0.2 eqv) and TEA (2 eqv) were allowed to react according to the general esierification procedure above in 100 mL of dry C¾C¾ for 24 h. The crude produc was purified by liquid chromatography on silica gel, elating with 95/5 DCM/MeOH increasing to 90/10 DCM/MeOH to give 22 as a light yellow/brown viscous oil. (77%) ^!H NMR

(400MHz, CDC1₃) 5.06 (s, br, NH), 3.15 (m, 4H), 2.68-2.35 (ni, 6H), 1.93 (s, 6H), 1.61 (q, 4H), 1.43 (s, 18H), 1.25 (d, 3H) °C NMR (IGQMHz, CDC1₃) 171 ,81 , 156.05, 79.57, 70.78, 59.62, 56.36, 38.65, 31.14, 30.17, 27.36, 18.26. m/z (ES MS) 510.2 [M-^;-Hf , 534.2 [M+Naf, 550.2 [M+K

Synthesis of 23 ~ 20, Bromoisobutyryl bromide (1.1 eqv.), DMAP (0.2 eqv) and

TEA (2 eqv) were allowed to react according to the general esterification procedure above in 100 mL of dry CH₂Cl₂ for 24 h. The crude product was purified by liquid chromatography on silica gel, eluting with 85: 15 CC¾ MeOH to give 23 as a brown viscous oil. (54%) Ή NMR (400MHz, CDC1₃) 4,92 (m, br, 2H), 3.63 (m, 1H), 3.37- 2.94 (m, 12! 11, 2.77-2.12 (m, 1 BH). 1.91 (s, 6H), 1.62 (m, 1.2H), 1 .44 (s₅ 36H), 1.20 m, 9H) m/z (ES MS) 1 168.7 [M+H^'f , 1192.7 [M+Na], 1208.7 [M+ ]⁺

Synthesis of Xant b (scheme 5) ~ Potassium ethyl xanthogenate (40,1 g, 250.2 rnmol) was transferred to a 500 mL two-necked round-bottomed flask, equipped with a magnetic stirrer bar, dropping funnel and septa cap with outlet. Acetone (150 mL) was added to the flask. 3-Bromopropionic acid (32.4 g, 211 .8 mmoi) was dissolved in acetone (80 mL) and transferred to dropping funnel. The acid was added to the flask dropwise with stirring. Once added, the reaction was left stirring at room temperature ovemighi. The initially yellow solid turns white as the reaction proceeds. The white solid is then filtered off and the solvent removed on the rotary evaporator. The resulting solid was dissolved in DCM (300 mL) and washed (1 x 200 mL distilled water and 2 x 200 mL brine). The organic layer was dried over MgSO,}, and the solid filtered off. The solvent was removed and placed in a vacuum oven to remove any residual solvent. Yield 59 %. ⁵H NMR (400 MHz, CDC1₃) δ: 1.42 (t, 3H), 2.85 (t₅ 2H)_> 3,38 (t, 2H), 4.63 (q, 2H)

Synthesis of Xant c (scheme 5) - Xanthate carboxy!ic acid, Xant b in (scheme 5) (15,0 g, 77.2 mmoi) was transferred to a 250 mL round-bottomed flask, equipped with a magnetic stirrer bar and sepia cap containing outlet. DCM (100 mL) was added. 5 drops of DMF was added. Oxa!yl chloride (19.6 g, 154.4 mmoi) was added dropwise via syringe with stirring, The reaction was left stirring for 2 hours. The reaction mixture changes from clear to a transparent orange as the reaction proceeds. The solvent was removed and washed twice with chloroform to remove any residual oxaiyl chloride. Resulting viscous orange oil used as obtained. Yield quantitative, ^'11 NMR (400 MHz, CDC1₃) δ: 1.42 ( 3H), 3.38 (m_s 4H), 4,63 (q, 2H).

Synthesis af Xmt d (scherae 5) - Bis-MPA (4.1 g, 30.9 mmoi), TEA (12.9 mL 101.2 mm i) and DMAP (188.6 mg, 1.6 mmoi) were transferred to a 250 mL two- necked) round-bottomed flask equipped with a magnetic stirrer bar, dropping funnel and septa cap containing outlet. The flask was then deoxygenated using nitrogen. Dry DCM (60 mL) was added via syringe under nitrogen. Xanthate acid chloride, Xant c in (scheme 5)... (16.4 g, 77.2 mmoi) was degassed with nitrogen inside the sealed dropping funnel. Dry DCM (10 mL) was added to dissolve the acid chloride. The xanthate acid chloride was added dropwise and the reaction was left stirring under nitrogen ovemiglit. The resulting solution was washed (1 x 200 mL distilled water and 2 x 200 mL brine). The organic layer was dried over MgSO^ and the solid filtered off. The solvent was reduced and the product, was ran through an automated flash column with a starting eluent of 95:5 hexane: ethyl acetate increasing to 20:80. Product fractions collected and solvent removed. The product was further washed with chloroform to remove residua! ethyl acetate, and solvent removed again.

Resulting oily product was placed in vacuum oven to remove any residua! solvent. Yield (35 ¾). ^lH NMR (400 MHz, CDC¾) δ: 1 .30 is, 3H), ,42 (t, 6H), 2,80 (t, 4H), 3.37 (L 4H). 4.30 (m, 4H), 4,65 (q, 4M).

Synthesis of Xant e (scheme S) - Xant d (scheme 5} (4,8 g, 9.9 mmol) was transferred to a 100 mL round-bottomed flask equipped with a magnetic stirrer bar and septa cap containing outlet. DCM (30 mL) was added, 5 drops of DMF were added. Oxalyl chloride (2.5 g, 19.8 mmol) was added dropwise via syringe. The reaction was left stirring for 3 hours. The solution changed from pale yello to dark orange as the reaction proceeds. The solvent was removed and the resulting oil was washed twice with chloroform to remove any residual oxalyl chloride. The product was in the form of viscous brown oil. Yield quantitative, i NMR. (400 MHz, CDC1₃) δ: 1 ,42 (m, 9H), 2.80 (t, 4H), 3.38 (t, 4H), 4,35 (m, 4H), 4.65 (q, 4H).

Synthesis of Xant-Gl (scheme 5)> Tertiary-bromoester alcohol (TBEA in scheme 5)(1.8 g, 8.6 mmol), TEA (1 .8 mL, 12.9 mmol) and DMAP (52.6 mg, 0.4 mmol) were transferred to a 100 mL two-necked round-bottomed flask, equipped with a magnetic stirrer bar, dropping funnel and septa cap containing outlet. The flask, was then deoxygenated using nitrogen. Dry DCM (30 mL) was added via syringe under nitrogen. Xant e (5.0 g, 9.9 mmol) was deoxygenated using nitrogen inside the sealed dropping funnel. Dry DCM (10 mL) was added via syringe. Xant e was added dropwise. The flask was cooled in an ice bath during this addition. The reaction was left stirring overnight. The resulting brown solution was washed (1 x 80 mL distilled water and 2 x 80 mL brine). The organic layer was dried over MgS0₄, and the solid filtered off. The solvent was reduced and the product was run through an automated flash column with a starting eluent of 100:0 hexane: ethyl acetate increasing to 20:80. Product fractions collected and solvent removed. The product was further washed with DCM to remove residual ethyl acetate, and solvent removed again. The resulting yellow/brown oil was left in a high vacuum vessel overnight to remove any residual solvent. Yield (40 %). ^!H NMR (400 MHz, CDC1₃) δ: 1.28 (s, 3H), 1.43 (t, 6H), L95 (s, 6H), 2.78 (t, 4H), 3.37 (t, 4H), 4.25 (m, 4H), 4.42 (m, 4H), 4.65 (q, 4H). Mass spec: m/z = 703.0 [M-i-Na .

1.4.6 GL G2. G3 Xanthate dendron synthesis using bisMPA backbone For key references relating to the synthesis ofbis-MPA dendrimers, refer to the following:

Macromolecuies 2002, 35, 8307-8314

J. Am. Ch m. Soc, 2001, 123, 5908-5917

J. Am. Chem. Soc, 2009, 131, 2906-2916

For preparation of benzylidene protected bis-MPA anhydride foiiow:

J. Am. Chem. Soc, 2001, 123, 5908-5917

For preparation of DPTS 4~(Dimethyiannno)pyridiniurn 4-toluenesu!fonate follow:

J. S. Moore, S.I. Stupp, Macromolecuies, 199Q, 23, 65

For preparation of 2-hydroxyethyl 2-bromo-2-methylpropanoate follow:

J. Mater. Chem., 201 1 ,21 , 1 8623-18629

Preparation ofXantkate based carbox lic acid building block:

Scheme / -Xanthate building Mock 1

Synthesis of 2-((Ethoxycarbonothioyl)thio)acetic acid 1 - A 500 mL round-bottomed flask equipped with a dropping funnel was charged with a magnetic stirrer bar, potassium ethyl xanthogenate (26.77 g₅ 167 mmol), and acetone (75 mL). A solution of 2-broffioacetic acid (19,31 g, 103 mmol) in acetone (40 mL) was added dropwise at room temperature over a period of 60 min. Stirring was continued overnight at room temperature. Solids were removed by filtration to afford a clear pale yellow solution. The solids on the funnel were washed with acetone (total of 50 mL). The combined washing and filtrate solutions were concentrated under vacuum to furnish a yellow viscous liquid that was dissolved in dichlorometliane (150 mL). This solution was washed twice with brine (100 mL), and the organic phase was dried over MgS04 and evaporated to dryness to afford 18.75 g (75%) of a white solid. Έ NMR. (400 MHz, CXX¾): δ - 1.43 ( J - 7,32 Hz, 3H), 3.98 (s, 2H) 4.67 (q, J - 7.25 Hz, 2H), 4.53 °C NMR (100 MHz, CDC1₃): δ ^::= 13.68, 37.60, 70.93, 174.30, 212.0!

Scheme 2 --- Preparation ofbis-mPA dendrom using anhydride chemistry

General procedure for dendon growth (2, 4 and 6) - To a SOOniL oven-dried round- bottom flask equipped with a magnetic stirrer (under nitrogen atmosphere), the henzylidene protected anhydride, the hydroxyl-te rninated dendron (generation 0 through to 3). and 4~dimethylaminopyridine (DMAP) were all dissolved in a 1 :1 ratio of CH2CI2 : pyridine (v/v). After stirring at room temperature for over 12 h, approximately 2 mL of water was added and the reaction was stirred for an additional 18 h in order to quench the excess anhydride. The product was isolated by diluting the mixture with CH2CI2 (150 mL) and washing with 1 M NaHS04 (3 x 150 ml,}, saturated aqueous NaHCOj (2 x 150 mL), and brine (150 mL). The organic layer was dried over MgS04 and evaporated to dryness. Any residual solvent was removed under high vacuum overnight to yield a white foam with a typical yield greater than 95%.

General procedure for deprotection of henzylidene by hydrogenation (3, 5 and 7) To a reactor suitable for medium pressure hydrogenation titled with a magnetic stirrer, the henzylidene protected dendrimer was dissolved in a 1 : 1 mixture of CH2CI2 : MeOFf (v/v). Pd(OH)2 on carbon (20%) was added and the reactor was evacuated and back-filled with hydrogen three times (¾ pressure : 10 bar). After vigorous stirring for 16 h, the reaction mixture was filtered through celite using a Buchner funnel and the filtrate was evaporated to dryness on a rotary evaporator under vacuo. The product was isolated as white foam in quantitative yields.

Scheme 3 - Preparation ofXanatkate dendrons and ATRP initiators

General procedure for surface group modification to Xanthates (8, 9 and 10} - To a

500mL oven-dried round-bottom flask equipped with a magnetic stirrer (under nitrogen atmosphere), the hydroxyl-terminated dendron (generation 0 through to 3), 2-((Ethoxycarbonothioyl)thio)acetic acid 1, and 4-(Dimethylamino)pyridinium 4- toiuenesulfonate (DPTS) were all dissolved in the minimum amount of CH₂CI₂. After the reaction flask was flushed with nitrogen, DCC was added. Stirring at room temperature was continued for 18 h under a nitrogen atmosphere. Once the reaction was complete the DCC- urea was filtered off and washed with a small volume of CH2CI2. The crude product was purified by liquid chromatography on silica gel, eluting with hexane gradually increasing to 40:60 ethyl acetate hexane to give a yellow viscous oil. General procedure for deprotection of para-toluene lfon l ester (TSe) by DBU (11, 12 and 13) ^■■ To an oven-dried round-bottom flask equipped with a magnetic stirrer, the benzylidene protected dendrimer was dissolved in 50 mL of CH₂G₂. 1.4mL of 1 ,8" diazabicyclo[5.4,0]imdeC"7-ene (DBU) was added. The reaction was stirred under a nitrogen atmosphere for 3hrs and monitored until completion by TLC (60:40 hexane:ethyl acetate). The product was isolated by diluting the mixture with Ο ¾ (100 mL) d washing with 1 M aHSC¼ (2 x 100 mL). The organic layer was dried over M.gS04 and evaporated to dryness. The product was then precipitated three times from hexanes. Any residual solvent was removed under high vacuum to yield a viscous oil with typical yields greater than 95%.

General procedure for focal point modification to an ATRP initiator by DCC/DPTS couplings (14, 15 and 16} - To a 500mL oven-dried round-bottom flask equipped with a magnetic stirrer (under nitrogen atmosphere), the carboxylic acid focal point xanthate dendron (generation 0 through to 3), 2-hydroxyethyi 2-bromo-2- methylpropanoate, and 4-(Dimethylamino)pyridinium 4-ioluenesulfonate (DPTS) were all dissolved in the minimum amount, of C¾C¾. After the reaction flask was flushed with nitrogen, DCC was added. Stirring at room temperature was continued for I S h under a nitrogen atmosphere. Once the reaction was complete the DCC- urea was filtered off and washed with a small volume of CH2CI2. The crude product, was purified by liquid chromatography on silica gel, eluting with hexane gradually increasing to 40:60 ethyl acetate/hexane to give a dark yellow viscous oil, Synthesis of 2 - The dendron growth step was carried out as described above, using para-toluene sulfonyl ethanol (lOg, 50 mmoi), benzyhdene anhydride (42.65 g, 100 mrnol, 2 equiv) and DMAP (2,57 g, 21 mmol)) dissolved in 220 mL of dry CH2CI2 and 120 m^'L of pyridine, and stirred for 16 h at room temperature. Yield: 19.78 g, white foam (98%). ^!H NMR (400 MHz, CDCI3): ft - 0.96 (s, 3H), 2.43 (s, 3H), 3.47 (t, J^{■■■■■■■} 6,3 Hz, 2H), 3.60 (d, J - 1 1.6 Hz, 2H) 4.47 (t, J = 6,26 Hz, 2H), 4,53 (d, J 11.54 Hz, 2H), 5.43 (s, 1H), 7.33 (m, 5H), 7.41 (m, 21:1), 7,81 (d₅ J - 8.42 Hz, 2H). ^t3C NMR. (100 MHz, CDCI₃): δ - 17.51, 21.64, 42,46, 55,13, 58.20, 73.32, 101.72, 126,15, 128.19, 128.23, 129.01 , 130.09, 136,01, 145.11, 149.86, 1 73.52, Synthesis of 3 ~ Deprotection of 2 (5.5g, 13,60 mmol) in 210 mL of CH₂Cl₂ : MeOH

(1 : 1 , v/v) was carried out as above for 16 h at room temperature under 10 bar Ha atmosphere. 0.55g Pd(OH)₂ was used. Yield: 4.3 g, white foam (99%). ^!H NMR (400 MHz, CDiOD): δ - 1 ,03 (s, 3H), 2.45 (s, 3H), 3.50 (dd, J - 42,53, 10.95 Hz, 4H), 3.59 (t, J - 5,98 Hz, 2H), 4.39 (t, J = 5.85 Hz, 2H), 7.47 (d, 2H), 7.82 (d, 2H), C NMR (100 MHz, CD₃OD): δ - 17.07, 21.61, 51.58, 55,90, 58,93, 65.66, 129.30, 1.31.22, 137.76, 146,71 , 175.89.

Synthesis of 4■· The dendron growth step was carried out as described above, using 3 (4.10g, 12.96 mmol), benzyhdene anhydride (16.58 g, 39 mmol, 3 equiv) and DMAP (0.7! g, 5.38 mmol)) ail dissolved in 70 mL of dry CH₂C1₂ and 35 mL of pyridine, and stirred for 16 h at room temperature. Yield: 8,68 g, white foam (94%), ¾ NMR (400 MHz, CDCI₃): § ^ 0.95 (s, 6H), 1.09 (s, 3H), 2.37 (s, 3H), 3.10 (t, J - S.8 Hz, 2H), 3.60 (d, J - 12.45 Hz, 4H) 4.20 (m, 6H), 4,56 (t, 1 === 9 Hz, 4H), 5.42 (s, 2H), 7,30 (m, 8H), 7.39 (m, 4H), 7.68 (d, i - 8.43 Hz, 2H). ^l3C NMR (100 MHz, CDC¾): δ = 17.33, 17.72, 21.56, 42.60, 46.70, 54.65, 58,32, 65.20, 73,46, 73.53, 101.63, 126.12, 128.05, 128,16, 128.91 , 130.00, 136.29, 137.78, 145.00, 172.00, 173.17. Accurate MS Cak'd for £½Η 4θ;₂8 [M + Naf - 747.2451. Found: [M + Naf = 742.2426, ES MS: [M + Naf ^::: 747.20, [ M + Κ Γ === 763.2 Synthesis of 5 - Deproiection of 4 (7.90g, 10,90 mniol) in 190 mL of CH2G₂ :

MeOH (1:1 , v/v) was carried out as above for 16 h at room, temperature under 10 bar H₂ atmosphere. 0.40g Pd(OH)₂ was used. Yield: 5.93 g, white foam (99%). ^lH NMR (400 MHz, CD₃OD): 6 - L15 (s, 9H), 2.48 (s, 3H), 3.57-3.69 (m, 10H), 4.1 1 (dd_s J - 31.18, 9.37 Hz) 4H), 4.46 (t, J - 5.77 Hz, 2H), 7.49 (d, J = 8.81 Hz, 2H), 7.85 (d, J - 8.39 Hz, 2H). °C NMR (100 MHz, CD₃OD): δ - 15.38, 15.94, 19.72, 45,76, 49.91, 53.92, 57.75, 63.95, 64.25, 127.40, 129.41 , 136.02, 144.82, 171.81 , 173.94. Accurate MS Calc'd for C ^OnS [M+Naf m z - 571.1825, [M + Naf m/z ~ 571.1 821, Found ES MS: [M + Naf - 571.2, [M + Kf - 587.2

Synthesis of 6 - ^'The dendron growth step was carried out as described above, using 5 (2.5g, 4.56 mmol), benzylidene anhydride ( i 1.67 g, 27.36 mmol, 6 equiv) and DMA? (0.35 g, 2.83 mmol)) all dissolved in 46 mL of dry CH₂CI₂ and 23 mL of pyridine, and stirred for 16 h at room temperature. Yield: 6.23 g, white foam (94%). 1H NMR (400 MHz, CDCI₃): δ - 0.93 (m, 15H), 1.19 (s, 6H), 2.39 (s, 3H), 3.28 (t, J ==== 6,38 Hz, 2H), 3.58 (d, J - 1 1.82 Hz, 8H), 3.94 (dd, J - 30.95, 11.33 Hz, 4H), 4.33 (m, 10H), 4,56 (d, .1 - 1.2 Hz, 8H), 5,40 (s, 4H), 7.30 (m, 14H), 7.39 (m, 8H), 7.74 (d, J = 8.52 Hz, 2H). °C NMR (100 MHz, CDCI₃): δ = 16.85, 17,66, 21.59, 42.59, 46.30, 46.87, 54.58, 58.22, 65.14, 65,70, 73.44, 73.52, 101.68, 126.20, 128.07,

128.13, 128.88, 130.04, 136.26, .137.82, 144.50, 171.63, 171.83, 173.20. ES MS: [M + Naf - 1387.5, [M + K === 1403.5

Synthesis of 7 - Deprotection of 6 (5.80g, 4.25 mmol) in 200 mL of (¾(¾ : MeOH (1 : 1, v/v) was carried out as above for 16 h at room temperature under 10 bar ¾ atmosphere. 0.29g Pd(OH)₂ was used. Yield: 4.3 Ϊ g, white foam (99%). *H NMR. (400 MHz, CD₃OD): δ - 1,15 (m, 15H), 1.28 (s, 6H), 2.48 (s, 3H), 3.62. (m, 18H), 4.24 (m, 12H), 4.48 (t, J - 6.14 Hz, 2H), 7.49 (d, I - 8.10 Hz, 2H), 7.85 (d, J = 8.19 Hz, 2H). ES MS: [M ·ί· Naf - 1035.4, [M + Kf - 1051.4

Synthesis of 8 - 1, 4.65g (25.80 mmol), and 2.72g (8.60 mmol) of 3, 1.01 g (3,44 ramol) of DPTS, and 5.86g (28.38 mmol) of DCC were allowed to react according to the genera! esierirkation procedure in 40 mL of dry CH₂CI₂ for 18 h. The crude product was purified by liquid chromatography on silica get, eluting with hexane gradually increasing to 40:60 ethyl acetate/hexane to give 6 as a yellow viscous oil 4.6g (84%). ^!'H NMR (400 MHz, CDC¾): δ === 1.16 (s, 3H), 1.42 (t, J = 7.15, 6H), 2.46 (s, 3H), 3.44 (t, J - 6.3 Hz, 2H), 3.91 (s, 4H), 4.18 (dd, J = 31.72, I 1 .36 Hz, 4H) 4.46 (t, J~ 6.03 Hz, 2H), 4.64 (q, 7.12 Hz, 4H), 7.39 (d, J - 8.23, 2H), 7.80 (d, J === 7.70, 2H). C NMR (100 MHz, CDC1₃): δ - 13.74, 17.56, 21.67, 37,70, 54.97, 58.36, 60.39, 66.21 , 70.91 , 128.12, 130.18, 136.18, 145.28, 167.33, 171.80, 212.57. ES MS: [M + Naf - 663.0, [M + Kf ==== 679.0

Synthesis of 9 - 1, 9.97g (55.32 mtnol), and 5.06g (9.22 mmol) of 5, 2.17g (7.38 mmol) of DPTS, and 1.2.56g (60.85 mmol) of DCC were allowed to react according to the general esterification procedure in 170 mL of dry Cl- Cl? for 18 h. The crude product was purified by liquid chromatography on silica gel, eluting with hexane gradually increasing to 50:50 ethyl acetate/hexane to give 6 as a orange viscous oil 9.65g (88%). Ή NMR (400 MHz, CDC1₃): δ - 1.20 (s, 3H), 1.25 (s, 6H), 1.42 (t, J = 7.16, 12H), 2.47 (s, 3H), 3.44 (t, J = 5.97 Hz, 2H), 3.94 (s, 8H), 4.25 (m, I2H) 4.46 (t, J^~ 5.90 Hz, 2H), 4.64 (q, J= 7.01 Hz, 8H), 7.40 (d, J ^« 8.51, 2H), 7.82 (d, J - 8.31, 2H).

Synthesis of 10 - See the general procedure

Synthesis of 11 -The removal of the para-toluene sulfonyl protecting group was carried out as described above, using 8 (4.60 g, 7.18 mmol, 1.0 equiv), and DBU ( 1.40mL, 9.33 mmol, 1.3 equiv) dissolved in 80 mL of CH₂Cl₂ and stirred for 3 h. The reaction was monitored using TLC, 40:60 ethyl acetate/hexane. Yield: 3.29 g, orange viscous oil (99%). ^!H NMR. (400 MHz, CDC1₃): δ = 1.32 (s, 3H), 1.42 (t, J ^« 7.05, 6H), 2.47 (s, 3H), 3.94 (s, 4H), 4.33 (dd, /- 39.96, 11.14 Hz, 2H), 4.64 (q, J = 7.14 Hz, 4H). ⁱ³C NMR (100 MHz, CDCI3): δ ^» 13.74, 17.86, 37.74, 46.06, 66.13, 70.87, 167.45, 177.80, 212.53. ES MS: [M. + Naf ^::: 481.0

For the synthesis of 12 and 13, - see the general procedure

1 , 1 '-Carbonyldiimi azoie (6.0994 g, 37,62 mmol) was added to a 2-tieck round bboottttoomm ffllaasskk,, wwhhiicchh mmaaggnneettiicc ssttiirrrriinngg,, ccoonnddeennsseerr aanndd aa >½ iinnlleett..

Anhydrous toluene (60 mi) and N-(2-Hydroxypropyl)morpholine, 1, (5,35 ml, 37,62 mmol) were added and the reaction was heated to 60 °C. The ABs brancher (3.5603 g. 18.81 mmol) dissolved in anhydrous toluene (6.0 nil) was added after 3 hours of reaction. After a further 16 hours the reaction was stopped, the toluene removed, in vacuo, the crude mixture was dissolved in dichloromethane (100 ml) and washed with NaOH solution (pH 14) (3 x 100 nil). The organic phase was dried over the solvent removed in vacuo and the resulting yellow oil was dried further under high vacuum to give 2, (75 %). "H NMR (400 MHz, CDCi₃): δ 1.13 (d, 3H), 1.22 (d, 6H), 1.67 (m, 4H), 2.25-2.65 (br m, 18H), 3.22 (m, 4H), 3.68 (m, 8H), 3.79 (m, I H), 4.98 (m, 2H), 5.29 and 5,40 (br s, H). ⁱ³C NMR (100 MHz, CDCI₃): δ 19.30, 20.83, 27.58, 27.76, 39.59, 52.28, 54.39, 62.86, 64.08, 67.36, 67.96, 68.12, 156.73. Calcd.: [Mf m/z = 531.36. Found: ES-MS: [M+Hf = 532.4, [M+Na]⁺ = 554.4. Found, C, 56.58; H, 9.24; N, 13.23 %. ¾H₄9N_S0₇ requires, C, 56.47; H, 9.29; N, 13.17 %,

2, (7.546 g, 14,2 mmol) was dissolved in DC (150 ml) and bubbled with N2 for 20 minutes. 4-(Dimethylamino)pyridme (DMAP) (86.7 nig, 0.7 mmol) and

triethylamine (TEA) (2.37 ml, 17.0 mmol) were added and the reaction vessel was cooled to 0 °C. a-Bronioisobutyryl bromide (1 ,93 mi, 15.6 mmol) was added dropwise, then the reaction was warmed to room temperature for 16 hours. The reaction colour changed from pale yello to a dark peach colour over this time period. The organic phase was washed with a saturated solution of NaHCOs (3 x 150 ml) and distilled water (3 x 150 n l), dried over NaiSC * and the solvent removed in vacuo to give a crude brown coloured oil. This was purified by silica column chromatography with a mobile phase of EtOAcrMeOH (4: 1), (Rf = 0.49) to give a light brown coloured oil, 3, (49 %). ^SH NMR (400 MHz, CDClj): δ 1.24 (m, 9H), 1 ,65 (m, 4H), 1.92 (d, 6H), 2.26-2.70 (br m, 18H), 3.20 (m, 4H), 3,69 (m, SH), 4.98 (m, 2H), 5.06 (m, 1H) 5.36 (br s₃ NH), ^UC NMR (100 MHz, CDC1₃): δ . Ca!cd,: [M}⁺ m/z = 679.32, Found: ES-MS: [M+H]⁺ = 680.3, [ ⁺-Na]⁺ == 702,3. Found, C, 50.87; H, 7.95; N, 10.37 %. CagH^NsOgBr requires, C, 51.17; H, 8.00; N, 10.29 ¾,

1 , -Carbonyidnmidazoie (9.729 g, 60.0 mmol) was weighed into a 3 -neck round bottom flask fitted with a N2 inlet, magnetic stirrer and condenser. Anhydrous THF (120 mi) was added via double ended needle. The reaction was heated to 60 °C and iPbisMPA (10.4514 g, 60.0 mmol) was added under a positive h½ flow. Reaction could be observed by the evolution of CO? and the reaction became effervescent. To avoid too much effervescence the iPbisMPA was added slowly, approx. 2g at a time once the effervescence had died down. After 3 hours the reaction mixture was bubbled through with !"½ to ensure any residual CC had been removed from the reaction medium and flask. The AB? brancher (5,949g, 30.0 mmol) was added dropwise in anhydrous THF (20 ml), after a further 18 hours the reaction was stopped and THF removed in vacuo. The crude residue was dissolved in DCM (125 ml) and washed with aOH solution (pH 14) (3 x 125 ml) and distilled water (125 nil). The organic phase was dried over the DCM was removed in vacuo then under high vacuum, to give a pale yellow oil, I, (78 %). Ή NMR (400 MHz, CDCI3); δ 1 ,02 (s, 6H), 1.10 (d, 3H), 1.42 (s, 6H), 1.47 (s, 6H), 1.70 (m, 4H), 2,32 (d of d of d, 2H), 2,45 (m, 2H), 2.63 (m_} 2H), 3.34 (q, 4FI), 3.75 (m, 5H), 3.92 (d, 4H). , ³C NMR (100 MHz, CDC1₃): δ 18.30, 19.1 1 , 20,38, 27.57, 29.08, 37.87, 40.59, 51.85, 63.00, 63.64, 67.54, 98.93, 175.24. Calcd.: [M]⁺ mfc = 501.34. Found: CI-MS:

[M+H]⁺= 502.7. Found, C, 59.86; H, 9.41 ; N, 8.18 %, C25H47N3G7 requires, C, 59,86: H, 9.44; N, 8,38 %. Gl MPA OH dendron (5.127 g, 10.2 mmol) was weighed into a round bottom flask and dissolved in DCM (70 nil) and degassed with dry nitrogen for 10 min. DMAP ( 62 mg, 0.51 mmol) and TEA (1 ,71 ml, 123 mmol) were added, the vessel was maintained under a positive nitrogen How and cooled to 0 °C. a-Bromoisobutyryl bromide (1.38 ml, 1 1.2 mmol) was added dropwise then was warmed to room temperature for 18 hours. The reaction was a light yellow colour to begin with and changed to a slightly darker yellow over time, no precipitate was observed. The reaction mixture was washed with a saturated NaHCQj solution (3 x 100 ml) and water (3 x 100 n l), dried over NaiSCX_? and concentrated in vacuo to give Gl MPA Br, 2, (54 %) as a yellow viscous oil. Ή NMR (400 MHz, CDClj): δ 1.04 (s, 6H), 1.24 (d, 3H), 1.42 (s, 6H), 1.46 (s, 6H), 1.67 (m, 4H), 1.91 (s, 6H), 2.40-2,67 (m, 6H), 3.31 (m, 4H). 3.74 (d, 4H), 3.96 (d, 4H), 5.05 (m, IH). ^{! 3}C NMR (100 MHz, CDClj): δ 18.35, 18.43, 27.58, 2.8.48, 31.16, 37.85, 40.69, 52.09, 56.54, 59.54, 67.44, 67.51 , 70.94, 98.74, 171.67, 175.12. Calcd.: [Mf m/z - 649.29. Found: ES-MS: [M+Hf === 650.3. Found, C, 53.61 ; H, 8.16; N, 6.42 %. requires, C, 53.53; H, 8.06; N, 6.46 %.

1.4.9 GI~A Terha jsrm^^ initiator

1 -dimethylamino-2~propanol (2.4758 g, 24 mmol, 4 eq.) was added to a 100 mL 2 necked round-bottomed flask containing anhydrous toluene (20 mL) and fitted with a reflux condenser, magnetic stirrer and a positive flow ofNi. The solution was stirred at room temperature and GDI (1.9458 g, 12 mmol, 2 eq.) was added. The mixture was heated to 60°C with stirring for 6 hours. AB? brancher (h i 358 g, 6 mmol, 1 eq.) dissolved in anhydrous toluene (5 mL) was deoxygenated using a N2 purge for 10 minutes and was added drop wise while the solution was stirred and the temperature was maintained at 6Q°C. The reaction was stirred for a further 18 hours at 60°C, and then allowed to cool to room temperature. The solution was concentrated in vacuo, and the remaining oil was dissolved in DCM (30 mL) and washed with 1M NaOH solution (3 x 30 mL). The solution was dried with anhydrous Na^SG, filtered and concentrated in vacuo to give Gl-A as a viscous liquid. Ή NMR (400 MHz, CDCI3) δ 1.25 (m, 9H), 1.64 (m, 3H), 2.05-2.67 (m, 22H), 3.20 (m, 3H), 3.78 (m, 1H), 4,89 (m, 2H). m/z (ES MS) 448.4 [M+H]+, 470.3 [M-^;-Na]+, Synthesis of Gl-A dendron initiator

Gl-A (0.8944 g. 2 mrnol, 1 eq. h TEA (0.2833 g, 2,8 mrnol, 1 ,4 eq.) and D AP (24.43 n g, 0.2 mrnol, 0.1 eq.) were added to a 100 mL 2 necked round-bottomed flask containing DCM (40 mL). The flask was deoxygenated under a positive N2 purge for 10 minutes, a-bromoisobutyryl bromide (0.4828, 0.26 mL, 2.7 mmol, 1,05 eq.) was added drop wise while the solution was stirring in an ice bath under a positive flow of N2. The reaction mixture was allowed to warm to room temperature and left stirring overnight. The organic phase was washed with saturated sodium hydrogen carbonate (NaHCOs) solution (3 30 mL). The solution was dried with anhydrous Na_?.S0₄, filtered and concentrated in vacuo to give initiator Gl-A as a viscous yellow liquid. ^fH NMR (400 MHz, CDC1.0 δ 1.24 (m, 9H), 1.64 (m, 4H), 1.92 (d of d, 8H), 2.05-2,05-2.67 (m, 22.H), 3,21 (m, 4H), 4.89 (m_f 2H), 5,06 (m, I H). m/z (ES MS) 596.3 [M+Hj+_? 617.3 [M+Na]+, 639.2 [M+ J-K

1.4.10 Gl-D Tertiarv amine dendron initiator Synthesis o/ G!-D dendron (HR2-B6)

2~{Dimethylamino)ethyi aerylate (6.0 g, 42 mmol, 6 eq.) was added to a 50 ml, round 2 necked round-bottomed flask containing IPA (12 mL). The flask was deoxygenated under a positive N_∑ purge for 10 minutes. I -arnino-2-propano! (0.5246 g, 7.0 mmol, 1 eq.) dissolved in IPA (12 mL) was added drop wise while the solution was stirring in an ice bath under a positive flow of N₂. The final mixture was stirred for a further 10 minutes at 0°C before being allowed to warm io room temperature and let! stirring for 48 hrs. The solvent was removed and the product left to dry in vacuo overnight. ^¾H NMR (400 MHz, CDC ) S 1 ,08 (d, 3H), 2.18-2.62 (m, 22H), 2.69 (rn, 2H), 2.89 (m, 2H), 3.77 (m, 1H), 4.16 (m, 4H). m/z (ES MS) 362.3 [M+H]+, 384.3 [M+Na]+.

Synthesis of G!-D dendron initiator (HR2-I43)

Gl-D dendron (1.1207 g, 10.86 mmol, 1 eq.), TEA (1 .5390 g, 15.2 mmol, 1.4 eq.) and B AP (132.7 mg, 1.086 mmol, 0.1 eq.) were added to a 250 mL 2 necked round-bottomed flask containing DCM (160 mL). The flask was deoxygenated under a positive N₂ purge for 10 minutes, a-bromoisobutyryl bromide (2.622 g, 1.4 mL, 1 1.4 mmoi, 1.05 eq.) was added drop wise while the solution was stirring in an ice bath under a positive flo of N₂. The reaction mixture was allowed to warm to room temperature and left stirring overnight. The organic phase was washed with saturated sodium hydrogen carbonate (NaHC(¼) solution (3 x 160 mL). The solution was dried with anhydrous Na₂SG and the product left to dry in vacuo overnight. ' H

NMR (400 MHz, CDC¾) δ 1.22 (d, 3H)₃ 1.89 (m, 6H), 2.24-2.69 (m, 22H), 2.83 (m, 4H), 4.20 (m, 41:1), 5.0 (m, 2H). m/z (ES MS) 510.2 [M+H]+, 534,2 [M+Na]+.

Synthesis o/G2~D dendron (HR2-116)

2~(Dimethylammo)ethy! acrylate (6,0 g, 42 mmoi, 6 eq.) was added to a 50 mL round 2 necked round-bottomed flask containing IP A (12 ml.,). The flask was deoxygenated under a positive N₂ purge for 10 minutes. Bis(3~

aminopropyl)amino)propan-2-ol (1.3221 g, 6.984 mmoi, 1 eq.) dissolved in IPA (12 mL) was added drop wise while the solution was stirring in an ice bath under a positive flow of N₂„. The final, mixture was stirred for a further 10 minutes at 0°C, allowed to warm to room temperature and left stirring for 48 hrs. The solvent was removed and the product left to dry in vacuo overnight. ^fH NMR (400 MHz. CDC ) δ 1.13 (d, 3H), 1.67 (m, 4H), 2.26-2.65 (m, 50H)_S 2.77 (m, 8H), 3,87 (m,lH), 4.17 (m, 8H). m/z (ES MS) 762.6 [M÷H]^~h 784.6 [M+Na]^~h -4.1 1 G2-D Tertiary amine dendron initiator

Synthesis of G2-D dendron initiator (HR2-12I)

G2-dendron (5.1431 g, 6.749 mmol, 1 eq.), TEA (0.9561 g, 9.449 mmol 1.4 eq.) and DMAP (82.5 rag, 0.6749 mmol, 0.1 eq.) were added to a 250 mL 2 necked round- bottomed flask containing DCM (160 mL). The flask was deoxygenated under a positive M₂ purge for 10 minutes, a-bromoisobutyryl bromide (1 ,629 g, 0.88 mL, 7.087 mmol, 1.05 eq.) was added drop wise while the solution was stirring in an ice bath under a positive flow of N . The reaction mixture was allowed to warm to room temperature and left stirring overnight. The organic phase was washed with saturated sodium hydrogen carbonate (NaliCO.;) solution (3 x 160 mL). The solution was dried with anhydrous Na?S0 and the product left to dry in vacuo overnight ^!H NMR (400 MHz, CDCI₃) δ 1.26 (d, 3H), 1.56 (m, 4H), 1.91 (m, 6H), 2.22-2.67 (m, 50H), 2,76 (m, 8H), 4.19 (m, 8H), 5,04 (m, 1H). m/z (ES MS) 912.5 | · ·! ]· 934.5 [M÷Na]+, 950.5 [M- K]+. 2. Pofydemira - 100% dendron initiated branched polymers

2.1.1.1 Aromatic dendrons GI and G2 DBOP Br

In a typical experiment, Gl BBOP Br (0.29 ! g, 0.69 mmoi) or GZ BBOP Br (0.648 g, 0.69 mmoi) and HP MA (targeted DP - 50) (5.0 g, 34.7 mmoi) were weighed into a round bottom flask. EGDMA (105 uL 0.55 mmoi) was added and the flask was equipped with magnetic stirrer bar, sealed and degassed by bubbling with b for 20 minutes and maintained under at 3(5 °C. Anhydrous methanol was degassed separately and subsequently added to the monomer/imtiator/brancher mixture via syringe to give a 50 % v/v mixture with respect to the monomer. The catalytic system; Cu(I)Cl (0,069 g, 0,69 mmoi) and 2_}2'~bipyridyl (bpy) (0,217 g, 1.39 mmoi), were added under a positive nitrogen flow in order to initiate the reaction. The polymerisations were stopped when conversions had reached over 98 %. The polymerisations were stopped by diluting with a large excess of tetrahydrofuran (THF), which caused a colour change from dark brown to a bright green colour. The catalytic system was removed using Dowex ^· Marathon ^! ' MSG (hydrogen form) ion exchange resin beads and basic alumina. The resulting polymer was isolated by precipitation from the minimum amount of THF into cold hexane. The

[initiator] :[CuCl]:[bpy] molar ratios in all polymerizations were 1 :1 :2. Other DPs targeted were DP20 and DP 100 with both Gl and G2 DBOP initiators.

2 L2 'BOC dendrons GI BOC Br

The Gl BOC Dendron initiator (1 OOmg, 0.186mmol) was added to a 25 mL round with a magnetic stirrer bar, followed by the addition of 2,2- bipyridyl (58Jmg, 0.372mmol), EGDMA (35.1mg, 0.177mmol) and HPMA (1.34& 9.28mmol). The reaction mixture was bubbled with N? for 15 minutes. Degassed anhydrous methanol (3.45mL) was added to the flask, and its contents stirred and bubbled with ]"¾ for a further 5 minutes. Copper (I) chloride (18.4mg, 0.1.86mmol) was quickly weighed out and added to the flask, instantly forming a brown coloured mixture, which was stirred and bubbled with -> for a further 5 minutes. A N? pressure was then built up within the flask, then N₂ nlet removed, and the flask stirred for 24 hours at 40 °C. Once the polymerisation was complete, THF was added to the reaction flask to poison the Cu (I) catalyst, forming a green coloured solution. The solution was passed through an alumina (neutral) column to remove the catalytic system, concentrated in vacuo, and precipitated into hexane. The supernatant was decanted off, and the remaining white solid dried overnight in a vac- oven, Xant-Gl initiator (578.0 mg, 0.868 mmol), BIPY (272.2 mg, 1.743 mmol), HP MA (6,3 g, 43.6 mmol,) and EGDMA (146.8 mg, 0,741 mmol) was transferred to 25 mL round-bottomed flask equipped with stirrer bar and sepia cap. The flask was deoxygenated using nitrogen. Separately deoxygenaied MeOH (12.9 mL, 38% w/v based on HPMA) added via syringe. Once all reactants had dissolved, nitrogen was bubbled through solution for 5 mins. Cu (I) CI (86.3 mg, 0.868 mmol,) quickly measured out and added to round-bottomed flask. Reaction mixture went from clear solution to deep red/brown. Nitrogen was bubbled through solution for an additional 10 mins. The reaction was then left to stir overnight under nitrogen, Reaction mixture forms a deep red/brown viscous liquid on completion. THF (20 mL) added to kill reaction. Once solution turned a bright, green colour, solution passed through a short alumina column to remove copper catalyst, yielding a translucent pale green solution. Solvent removed and resulting oily liquid precipitated into cold hexane (approx. 50 mL, cooled in dry ice bath). The resulting pale green crystals were filtered off and washed with cold hexane. The sample was placed in a vacuum oven to remove any residual solvent. 2.1.2.1 Gl-A Tertiary amine initiator

In a typical synthesis, targeting a number average degree of polymerisation (DP„) ^:::: 50 monomer units (poly(HPMA)so; DEAE . 'ni tiator: 50), bpy (173.3 mg, 1.1096 mmo , 2 eq.)_s HPMA (4 g, 27.7 rnmol, 50 eq.), EGDMA (77.0 mg, 0.3883 mmol, 0.7 eq) and isopropanol (IPA) (38.9% v/v based on HPMA) were placed into a 25 mL round-bottomed flask. The solution was stirred and deoxygenated using a nitrogen (H ) purge tor 15 minutes. Cu(i)Ci (54.9 mg, 0.5548 mmol, 1 eq.) was added to the flask and left to purge for a further 5 minutes. Gl-A dendron initiator (0.3310 g, 0.5548 rnmol, 1 eq.) was added to the flask under a positive flow of N₂, and the solution was left to polymerise at 40°C. Reactions were terminated when >99% conversion was reached, as judged by ^lH NMll, by exposure to oxygen and addition of THF. The catalyst residues were removed by passing the mixture over a basic alumina column. THF was removed under vacuum to concentrate the sample before precipitation into hexane and drying in the vacuum oven overnight.

Gl ML Br (0.378 g, 0.55 ramol) and HPMA (4.0 g, 27.7 mmol) were weighed into a round bottom flask. EGDMA (73.2 μΐ, 0.39 mmol) was added and the flask was equipped, with magnetic stirrer bar, sealed and degassed by bubbling with N₂ for 20 minutes and maintained under N_¾ at 30 °C. isopropan l was degassed separately and. subsequently added to the monomer/initiator brancher mixture via syringe to give a 50 wt wt% mixture with respect to the monomer. The catalytic system; Cu(l)Cl (0.055 g, 0.55 mmol) and 2,2'-bipyridyi (bpy) (0.173 g, 1.1 mmol), were added under a positive nitrogen flow in order to initiate the reaction. The polymerisations were stopped when conversions had reached over 98 %. The polymerisations were stopped by diluting with a large excess of tetrahydrofuran (THF), which caused a colour change from dark brown to a bright green colour. The catalytic system was removed using Dowex^® Marathon ^lhA MSG (hydrogen form) ion exchange resin beads and basic alumina. The resulting polymer was isolated by precipitation from the minimum amount of THF into cold hexane. The [initiator] :[CuCl]: [bpy] molar ratios in all polymerizations were 1 : 1 :2 2,1.2.3 GI bisMPA initiator

GI MPA Br (0.451 g, 0.69 mmol) and HPMA (5.0 g, 34.7 mmol) were weighed into a round bottom flask. EGDMA (1.05 μΐ, 0.55 mmol) was added and the flask was equipped with magnetic stirrer bar, sealed and degassed by bubbling with N-? for 20 minutes and maintained under ?, at 30 °C. Isopropanol was degassed separately and subsequently added to the monomer/iniiiator brancher mixture via syringe to give a 50 wt wt% mixture with respect to the monomer. The catalytic system; Cu(I)CI (0.0687 g, 0.69 mmol) and 2₅2^*-bipyridyl (bpy) (0.21.7 g, 1.39 mmol), were added under a positive nitrogen flow in order to initiate the reaction. The polymerisations were stopped when conversions had reached over 98 %. The polymerisations were stopped by diluting with a large excess of tetrahydrofuran (THF). which caused a colour change from dark brown to a bright green colour. The catalytic system was removed using Dowexⁱ Marathon™ MSG (hydrogen form) ion exchange resin beads and basic alumina. The resulting polymer was isolated by precipitation from the minimum amount ofTHF into cold hexane. The [miiiator]:[CnCi]:[bpy] molar ratios in all polymerizations were 1 :1 :2.

2.2.1 Gl-A Tertiary.amine dendron initiator in a typical synthesis, targeting a number average degree of polymerisation (DP„) ^::: 50 monomer units (poly(iBuMA)so; noEAE A/'abitiatcr: 50), bpy (175.7 mg, 1.1252 mmol, 2 eq.), iBuMA (4 g, 28.13 mmol, 50 eq.), EGDMA (105.9 mg, 0.5345 mmol, 0.95 eq) and aqueous isopropanol (7.5% water by volume) (33.3% v/v based on rBuMA) were placed into a 25 ml. round-bottoraed flask. The solution was stirred and deoxygenated using a nitrogen (N₂) purge for 15 minutes. Cu(i)Cl (55.7 mg, 0.5626 mmol, 1 eq.) was added to the flask and left to purge for a further 5 minutes. Gl-A dendron initiator (0.3356 g, 0.5626 mmol, 1 eq.) was added to the flask, under a positive flow of N₂, and the solution was left to polymerise at 40°C. Reactions were terminated when >99% conversion was reached, as judged by ^l.H NM , by exposure to oxygen and addition of THF. The catalyst residues were removed by passing the mixture over a basic alumina column, THF was removed under vacuum to concentrate the sample before precipitation into liexane and drying in the vacuum oven overnight

In a typical synthesis, targeting a number average degree of polymerisation (DP,,) = 50 monomer units (poly(DEAEMA)so; nos_A& ¾mtkior- 50), bpy (134,9 mg, 0.8637 rn.rn.oL 2 eq,), DEAEMA (4 g, 21 ,59 mmol, 50 eq,), EGDMA (77.0 mg, 0.3886 mmol, 0.9 eq) and IP A³' (38.9% v v based on DEAEMA) were placed into a 25 mL round-bottomed flask. The solution was stirred and deoxygenated using a Nj purge for 15 minutes, CuQG (42.8 mg, 0,4318 mmol, 1 eq.) was added to the flask and left to purge for a further 5 minutes. Gl -A dendron initiator (0.2576 g, 0,4318 mmol, 1 eq.) was added to the flask under a positive flow of j, and the solution was left to polymerise at 40°C. Reactions were terminated when >99% conversion was reached, as judged by ^!H NMR, by exposure to oxygen and addition of acetone. The catalyst- residues were removed by passing the mixture over a basic alumina column. Acetone was removed under vacuum to concentrate the sample before precipitation into cold petroleum ether (4Q°C - 60°C). The polymerisation conditions and procedure is identical to those described for linear polymers above and drying in the vacuum oven overnight,

2.3,2 GQ-P Tertiary amine dendron initiator

In a typical synthesis, targeting a number average degree of polymerisation (DP„) ^::: 50 monomer units 50), bpy (134.9 mg, 0,8637 mmol, 2 eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.), EGDMA (77.0 mg, 0.3886 mmol, 0.9 eq) and IPA³⁷ (38,9% v/v based on DEAEMA) were placed into a 25 ml, round-bottomed flask. The solution was siirred and deoxygenated using a h½ purge for 15 minutes. CuQCl (42,8 mg, 0.4318 mmol, I eq.) was added to the flask and left to purge for a further 5 minutes. GO-D dendron initiator (0.1089 g, 0,4318 mmol, 1 eq.) was added to the flask under a positive flow of N2, and the solution was left to polymerise at 40°C. Reactions were terminated when >99% conversion was reached, as judged by ^!H NMR, by exposure to oxygen and addition of acetone. The catalyst residues were removed by passing fee mixture over a basic alumina column. Acetone was removed under vacuum to concentrate the sample before precipitation into cold petroleum ether (40°C - 60°C) and drying in the vacuum oven overnight. The polymerisation conditions and procedure is identical to those described for linear polymers above.

In a typical synthesis, targeting a number average degree of polymerisation (DP„) ~ 50 monomer units (poly(DEAEMA)5o oEAE atnitiaior: 50), bpy (134,9 mg, 0.8637 mmol, 2 eq.), DEAEMA (4 g, 21 ,59 mmol, 50 eq.), EG DMA (77.0 mg, 0.3886 mmol, 0.9 eq) and IPA^J' (38,9% v/v based on DEAEMA) were placed into a 25 niL round-bottomed flask. The solution was stirred and deoxygenated using a Ν·> purge for 15 minutes. CuQCl (42.8 mg, 0.4318 mmol, I eq.) was added to the flask and left to purge for a further 5 minutes. Gl-D dendron initiator (0.2204 g. 0.4318 mmoi, 1 eq.) was added to the flask under a positive flow of N^, and the solution was left to polymerise at 40°C. Reactions were terminated when >99% conversion was reached, as judged by Ή NMR, by exposure to oxygen and addition of acetone. The catalyst residues were removed by passing the mixture over a basic alumina column. Acetone was removed under vacuum to concentrate the sample before precipitation into eoid petroleum ether (40^UC - 60^WC) and drying in the vacuum oven overnight. The polymerisation conditions and procedure is identical to those described for linear polymers above.

2,3,4 G2~B Te tiju^^

In a typical synthesis, targeting a number average degree of polymerisation (DP_«) 50 monomer units (poly(DEAEMA)5o; nmAmtJniasimQr'- 50), bpy (134.9 mg, 0,8637 mmol, 2 eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.), EGDMA (77,0 mg, 0.3886 mmol, 0,9 eq) and IP A³' (38.9% v/v based on DEAEMA) were placed into a 25 mL round-bottomed fiask. The solution was stirred and deoxygenated using a ]¾ purge for 15 minutes. CuQCi (42.8 mg, 0,4318 mmol, 1 eq.) was added to the flask and left to purge for a further 5 minutes. G2-D dendron initiator (0.3934 g, 0.4318 mmol, 1 eq.) was added to the flask under a positive flow of N , and the solution was left to polymerise at 40°C. Reactions were terminated when >99% conversion was reached, as judged by ^JH NMR, by exposure to oxygen and addition of acetone. The catalyst residues were removed by passing the mixture over a basic alumina column. Acetone was removed under vacuum to concentrate the sample before precipitation into cold petroleum ether (40°C - 60°C) and drying in the vacuum oven overnight. The polymerisation conditions and procedure is identical to those described for linear polymers above.

in a typical synthesis, targeting a number average degree of polymerisation (DP„) ^:::: 50 monomer units (poly(OEGMA)5o; DEAEMA ninitiatop 50), bpy (83,3 mg, 0,5333 mmol, 2 eq.), OEGMA (4 g, 13.3 mmol, 50 eq.), EGDMA (50.2 mg, 0.2533 mmol, 0.95 eq) and aqueous isopropanol (7.5% water by volume) (33.3% v/v based on OEGMA) were placed into a 25 mL round-bottomed flask. The solution was stirred and deoxygenated using a nitrogen (N₂) purge for 15 minutes. Cu(t)Cl (26.4 mg, 0.2667 mmol, 1 eq.) was added to the flask and left to purge for a further 5 minutes. Gl-A dendron initiator (0,1591 g, 0.2667 mmol, 1 eq.) was added to the flask under a positive flow of^'Nj, and the solution was left to polymerise at 40°C. Reactions were terminated when >99% conversion was reached, as judged by H NMR, by exposure to oxygen and addition of THF. The catalyst residues were removed by passing the mixture over a basic alumina column. THF was removed under vacuum to concentrate the sample before precipitation into cold hexane and drying in the vacuum oven overnight. In a typical synthesis, targeting a number average degree of polymerisation (DP,,) = 50 monomer units (po!y(DEAEMA)so; DEABtfA »iRi ator: 50), bpy (134,9 mg, 0.8637 tnmol, 2 eq,), DEAEMA (4 g, 21.59 mmol, 50 eq.) and isopropanoi (IPA) (37.7% v/v based on DEAEMA) were placed into a 50 niL round-bottomed flask. The solution was stirred and deoxygenated using a nitrogen N₂) purge for 15 minutes. Cu(j)CI (42,8 mg, 0.4318 mmol, 1 eq.) was added to the flask and left to purge for a further 5 minutes. G2-D dendron initiator (0.3934 g, 0,4318 mmol, i eq.) was added to the flask under a positive flow of N?, ami the solution was left to polymerise at 40°C. In another 25 mL round-bottomed flask, bpy (134,9 mg, 0.8637 mmol), iBuMA (4.0 g, 28, 1 mmol, 65 eq.), EGDMA (77.0 mg, 0.3886 mmol, 0.9 eq) and aqueous

isopropanoi (23.8% v/v based on iBuMA) were added. The solution was stirred and deoxygenated vising a nitrogen ( ₂) purge for 15 minutes, Cu(i)Cl (42.8 mg, 0,4318 mmol, 1 eq.) was added to the flask and left to purge for a further 5 minutes. After the conversion of DEAEMA reached around 85%, the inixture from the second flask was added into the first flask rapidly using a syringe and taking care not to admit any air into the vessel. A sample was taken immediately after the addition of the iBuMA monomer solution for ^!H NMR analysis. The block copolymerization reaction was carried out at ambient temperature and samples were taken periodically from the reaction mixture for Ή NMR analysis. Reactions were terminated when >99% conversion was reached, as judged by ' H NMR, by exposure to oxygen and addition of acetone. The catalyst residues were removed by passing the mixture over a basic alumina column. Acetone was removed under vacuum to concentrate the sample before precipitation into cold petroleum ether (40°C - 60°C) and drying in the vacuum oven overnight.

I fWe 1 - 100 % Dendron initiated pofydendrons

Generatio Initiator Polymer EGDMA

Mn (gmol^"') Mw (gmoi^{' s}) PDI n Functionality Core (mol%)

Gl DBOP pHPMA20 0.8 52 800 545 000

Gl DBOP pHPMA50 0,8 47 200 1 169 000 Gl DBOF pHPMAiOO 0,8 69 300 1 354 500 19,54

G2 DBOP pHPMA20 0.8 .153 000 1 565 000 10.23

G2 DBOF pHPMASO 0.8 59978 739440 12,33

G2 DBOP pHPMAiOO 0.8 164 200 2 227 500 13.58

G i tBOC pHPMASO 0,95 ¾ 45539 3.71

Gl X&mihafc pHPMASO 0.85 63800 1070000 15

Gl Morphoiine pHPMASO 0.7 76687 454746 5.93

Gl bisMPA pHPMASO 0.8 77745 436461 5.61

Gl-A f- amine pHPMASO 0,7 661 180 966552 1.50

Gi-A i-amine piB MASO 0.95 150264 284002 1.90

G i - A i-amine 0.9 201497 244622 1.20

0

Gl -A i'-amine pOEGMASO 0.95 97082 216813 2.20 pDEAEMAS

GO-D i-amine 0.9

0

Gl -D t~ am ne 0.9

0

pDEAEMAS

G2-D i-amine 0,9 125652 302557 2.40

0

pDEAEMAS

Q-b-

G2-D /-amine 0,9 129737 374192 2,90

?BuMA65- st~EGD.MA

?. Mixed initiator systems

3.1 Mixed de&dro&s

The Gi BOC Dendron initiator (67. 'BOC Dendron nitiator (63.1mg, 0.054mmol) was added to a 25 mL round bottom flask with a magnetic stirrer bar, followed by the addition of 2,2-bipyridyl (56.2mg, 0.360mmol), EGD A (28.5mg, Q.144mmol) and HPMA (1.3g, 9.0mmoi). The reaction mixture was bubbled with N₂ for 15 minutes. Degassed anhydrous methanol (3.3mL) was added to the flask, and its contents stirred and bubbled with N_∑ for a further 5 minutes. Copper (I) chloride (!7.8mg, O.lSOmraol) was quickly weighed out and added to the flask, instantly forming a brown coloured mixture, which was stirred and bubbled with N₂ for a further 5 minutes. A N? pressure was built up within the flask, then N? inlet then removed, and the flask stirred for 24 hours at 40 °C. Once the polymerisation was complete, THF was added to the reaction flask to poison the Cu (I) catalyst, forming a green coloured solution. The solution was passed through an alumina (neutral) column to remove the catalytic system, concentrated in vacuo, and precipitated into hexane, The supernatant was decanted off, and the remaining white solid dried overnight in a vac-oven.

3.2 Mixed d¾ndro¾ with . »o» deH.droa .iaitiai r

3.2.1 G2

In a typical reaction, G2 DBOP Br (0.259 g, 0.28 mmol) and 750 PEG initiator (0.250 g, 0.28 mmol) (for a targeted ratio of G2 dendron:750 PEG of 50:50 mol%) were weighed into a round bottom flask, followed by HPMA (4.0 g, 27.7 mmol). EGDMA (84 μΐ, 0.44 mmol) was added and the flask was equipped with magnetic stirrer bar, sealed and de assed by bubbling with Ν·? for 20 minutes and maintained under N₂ at 30 °C. Anhydrous methanol was degassed separately and subsequently added to the monomer/iniiiator/brancher mixture via syringe to give a 50 wt wt% mixture with respect to the monomer. The catalytic system; Cu(I)Cl (0.055 g, 0.55 mmol) and 2,2'-bipyridyl (bpy) (0.173 g, 1.1 mmol), were added under a positive nitrogen flow in order to initiate the reaction. The polymerisations were stopped when conversions had reached over 98 %. The polymerisations were stopped by diluting with a large excess of tetrahydrofuran (THF), which caused a colour change from dark brown to a bright green colour. The catalytic system was removed using Dowex* Marathon™ MSG (hydrogen form) ion exchange resin beads and basic polymer was isolated by precipitation from the minimum amount of THF into cold hexane. The [initiator] : [CuCl] : [bpy] molar ratios in ail polymerizations were 1 : 1 :2.

3.2.2 G2 DBOP Bnmd 2K H

In a typical reaction, G2 DBOP Br (0.324 g, 0.35 mmol) and 2K PEG initiator (0.745 g, 0.35 mmol) (for a targeted ratio of G2 dendron:750 PEG of 50:50 mol%) were weighed into a round bottom flask, followed by HPMA (5.0 g, 34.7 mmol). EGDMA (1 12 μΐ, 0.59 mmol) was added and the flask was equipped with magnetic stirrer bar, sealed and degassed by bubbling with a for 20 minutes and maintained under 1\½ at 30 °C. Anhydrous methanol was degassed separately and subsequently added to the monomer/initiator brancher mixture via syringe to give a 50 % v/v mixture with respect to the monomer. The catalytic system; Cii(I)Ci (0.069 g, 0.69 mmol) and 2,2'~bipyridyl (bpy) (0.217 g, 1.39 mmol), were added under a positive nitrogen flow in order to initiate the reaction. The polymerisations were stopped when conversions had reached over 98 %. The polymerisations were stopped by diluting with a large excess of tetrahydrofuran (THF), which caused a colour change from dark brown to a bright green colour. The catalytic system was removed using Dowex^® Marathon ^{l M} MSC (hydrogen form) ion exchange resin beads and basic alumina. The resulting polymer was isolated by precipitation from the minimum amount of THF into cold hexane. The [initiator] : [CuCl] : [bpy] molar ratios in all polymerizations were 1 : 1 :2.

3.2.3 Gl tBOC dendron and Lactose initiated pHPMA core The Gl ¾OC Dendron initiator (48.5mg, 0.0 mmol) and Lactose ATRP

initiator (70.7mg, 0.09mmol) was added to a 25 mL round bottom flask equipped with a magnetic stirrer bar, followed by the addition of 2,2-bipyridy! (56.2mg, 0.360mmol), EGDMA. (28.Smg_? 0, ! 44mmol) and HPMA (1.3g. 9.0mmol). The reaction mixture was bubbled with Nj for 15 minutes. Degassed anhydrous methanol (3.3mL) was added to the flask, and its contents stirred and bubbled with N2 for a further 5 minutes. Copper (I) chloride (17.8mg, O.l SOmmol) was quickly weighed out and added to the flask, instantly forming a brown coloured mixture, which was stirred and bubbled with N2 for a -further 5 minutes. A 2 pressure was built up within the flask, then N_: inlet then removed, and the flask stirred for 24 hours at 40 °C. Once the polymerisation was complete, THF was added to the reaction flask to poison the Cu (Ϊ) catalyst, forming a. green coloured solution. The solution was passed through an alumina (neutral) column to remove the catalytic system, concentrated in vacuo, and precipitated into hexane. The supernatant was decanted off, and the remaining white solid dried overnight in a vac-oven,

3.2.4 G 1 IBOC dendron and Afunctional .initiator pHPMA dumbbell core

The G1 ^'HOC Dendron initiator (I Slmg, 0.336mmol) and hi -· functional initiator (36,6mg, 0.084mmol) was added to a 25 mL round bottom flask equipped with a magnetic stirrer bar, followed by the addition of 2,2-bipyridyl (I 57.4mg, 1.Olmmoi), EGDMA (79.1mg, Q.399mmol) and HPMA (3.63g, 25.2mmol), The reaction mixture was then bubbled with N? for 15 minutes. Degassed anhydrous methanol (l GmL) was added to the flask, and its contents stirred and bubbled with Ν·> for a further 5 minutes. Copper (I) chloride (49.9mg, 0.504mmol) was quickly weighed out and added to the flask, instantly forming a brown coloured mixture, which was stirred and bubbled with ? tor a further 5 minutes. A N2 pressure was built up within the flask, then _¾ inlet then removed, and the flask stirred for 24 hours at 40 "C. Once the polymerisa ion was complete, THF was added to the reaction flask to poison the Cu (I) catalyst, forming a green coloured solution. The solution was passed through an alumina (neutral) column to remove the catalytic system, concentrated in vacuo, and precipitated into hexane. The supernatant was decanted off, and the remaining white solid dried overnight in a vac-oven.

3.2.5 G^jBOC gndrpiLasd Afunctional initiator pHPMA dumbbell mm

The G2 ¾€)€ Dendron initiator (197mg, 0.168mmoi) and bi-functional initiator (i S.3mg, 0.042mmol) was added to a 25 mL round bottom flask equipped with a magnetic stirrer bar, followed by the addition of 2,2-bipyridyl (78.7mg, 0.5G4mmol), EGDMA (33.3mg, 0.168mmol) and HPMA (3.63g, 12.6mmol). The reaction mixture was bubbled with N_¾ for 15 minutes. Degassed anhydrous methanol

(4.65mL) was added to the flask, and its contents stirred and bubbled with N? for a further 5 minutes. Copper (I) chloride (24.9mg, 0.252mmol) was quickly weighed out and added to the flask, instantly forming a. brown coloured mixture, which was stirred and bubbled with N_?. for a further 5 minutes, A N pressure was built up within the flask, then N₂ inlet then removed, and the flask stirred for 24 hours at 40 °C. Once the polymerisation was complete, THE was added to the reaction flask to poison the Cu (I) catalyst, forming a green coloured solution. The solution was passed through an alumina (neutral) column to remove the catalytic system,

concentrated in vacuo, and precipitated into hexane. The supernatant was decanted off, and the remaining white solid dried overnight in a vac-oven.

Table 2 - Mixed initiator polydendrons

EGDM

Polymer

Initiator 1 Initiator 2 A MB (gssoi ) Mw (grnof⁵! 1 PDI

Core

(moi%)

Gl iBOC G2 tBOC pHPMASO 0,8 61500 153500 2.49

G1 tBOC Lactose pHPMASO 0,8 102000 216000 2.11

Gl iBOC Afunctional pHPMASO 0.95 47000 227000 4.83

G2 tBOC biftmctional pHFMASO 0.8 177500 555500 3.13

G2 .D5QP 7,50 PEG

100 0 pHPMASO 0.8 90 500 1 304 000 9.67

90 10 pHPMASO 0.8 68457 1495000 21.84

75 25 pHPMASO o.s 5243 ! 987762 18.88

50 50 pHPMASO 0.8 39447 480638 12.19

25 75 pHPMASO 0.8 36157 315320 8.73

10 90 pHPMASO 0.8 37672 286049 7.61

0 100 pHPMASO 0.8 68133 296179 4.35

25 75 pHPMASO 0.9 60738 6751 19 11.13

0 100 pHPMASO 0.95 74740 642728 8.60

G2 DBOP 2K PEG

100 0 pHPMASO 0.8 193576 277^000 1 1 ,49

90 10 pHPMASO 0.8 348067 2464000 7.08 25 pHPMASO 0.8 55050 1067000 1938

50 pHPMASO 0.85 29372 709209 24.15

75 pHPMASO 0.95 141272 1862000 13.18

90 pHPMASO 0.95 40195 795274 19.79

100 pHPMASO 0.95 32246 476990 14.79

50 pHPMAIOO 0.8 79448 516794 6.51

4. Nanoprecipitation of Pofydendnms

4.1 NanpBgrticle formation (slow addition) - HR method

In a typical procedure, 10 mg of sample was completely dissolved in 2 mL. of acetone at room temperature; the resulting solution (5 mg mL^"1) was added drop wis* to 10 mL of distilled water under vigorous stirring for ca, 15 min using a glass pipette. The solution was stirred vigorously for 24 h at room temperature, until the acetone was completely evaporated as determined by Ή NMR analysis, where no peak at δ 2.22 corresponding to acetone was observed.

4.2 Nanoprecipitation (fast addition), Polydendrons were dissolved in ^'THF for a minimum of 6 hours at various

concentrations. Once fully dissolved polymer in THF (1 ml, 5 mg/ml) was added quickly to a vial of water (5 ml) stirring at 30 °C. The solvent was allowed to evaporate overnight in a fume cupboard to give a final concentration of 1 mg/ml polymer in water. By adjusting the starting concentration and the volume of water used, the size of the corresponding nanoparticles can be controlled to an extent. The nanoparticles .formed were analysed by dynamic light scattering (DLS) and

fluorimetry.

Table 3 - DLS data for 100% Dendron initiated polydendrons

^{' "} EGD A

Initiator Polvmer core Size (d.nm Ρϊ>1

Gl DBOP pHP A2G 0.8 61.72 0.1 17 Gi DBOP pHPMASO 0.8 63.9 0.130

Gl DBOP pHPMAlOO 0.8 69.89 0.070

G2 DBOP pHPMA20 0.8 81.33 0.076

G2 DBOP pHPMASO 0,8 80.78 0.083

G2 DBOP pHPMA!OO 0.8 80.56 0.1 19

Gl -A famine pHPMASO 0.7 70.6 0.366

Gi-A iamine piBuMASO 0,95 45.98 0.217

GI-A famine pDEAEMASO 0.9 1.36.2 0.148

Gl-A amine pOEGMASO 0.95 44.98 0.519

GO-D iamine pDEAEMASO 0.9

GI -D /famine pDEAEMASO 0.9

G2-D iamine pDEAEMASO 0.9 1 1 5,9 0, 158 pDEAEMA50-W<?c/e-

G2-D iamine 0.9 162.9

(BvMA-st-EGDMA

Xant Gl - post . modified

wife;

benzyl pHPMASO 0.85 141.1 0.238 n morphoHno pHPMASO 0,85 159.3 0,166

PEG480 pHPMASO 0.85 106.9 0.257

PEGSOOO pHPMASO 0.8S Ϊ 56.8 0.427

Table 4 -^■ DLS data for mixed initiator polydendro: EiS

Polymer EGDMA

Initiator 1 Initiator 2 Size (d. .»m) PDI

core (M0l%)

biftmciiona

Gl iBOC pHPMASO 0.95 3 , h 0.109

1

bifunctiona

G2 iBOC pHPMASO 0.8 27.33 0.1 16

1

G2 DBOP 750 PEG

100 0 pHPMASO 0.8 80.78 0.083

90 10 pHPMASO 0.8 1 15.6 0.069

75 25 pHPMASO 0.8 109.8 0.073

50 50 pHPMASO 0.8 114.6 0,067 pHPMASO 0,8 92.57 0.078

10 90 pHPMASO 0.8 94.26 0.091

0 100 pHPMASO 0.8 87.8 0.076

0 100 pHPMASO 0.95 89.53 0.083

100 0 pHPMASO 0.8 62.1 5 0.391

90 10 pHPMASO O.S 144.4 0.036 75 25 pHPMASO 0.8 214,6 0.085

50 50 pHPMASO 0.85 105.5 0.058

25 75 pHPMASO 0.95 52.17 0.277

10 90 pHPMASO 0.95 37.81 0.207

0 100 pHPMASO 0.95 36.18 0.24

50 50 pHPMA20 0.85 54.9 0.296

pHPMA! O

50 50 0.8 232,2 0.133

5.1 Nile Red :

In a typical procedure, 10 mg of sample and 0.1 mg ile Red was dissolved completely in 2 mL of acetone at room temperature; the resulting solution (5,05 mg mL^"f) was added drop wise to 10 mL of distilled water under vigorous stirring for ca. 15 min using a glass pipette. The solution was stirred vigorously for 24 h at room temperature, until the acetone was completely evaporated as determined by ^lH NM analysis, where no peak at 8 2.22 corresponding to acetone was observed.

5.2 Fluoresceinamine encapsulation - HR. method

In a typical procedure, 10 mg of sample and 1 mg of fluoresceinamine was dissolved completely in 2 mL of acetone at room temperature; the resulting solution (5.5 mg mL^"1) was added drop wise to 10 mL of distilled water under vigorous stirring for ca. using a glass pipette. The solution was stirred vigorously for 24 h at ; temperature, until the acetone was completely evaporated as determined by Ή NMR analysis, where no peak at δ 2.22 corresponding to acetone was observed.

5.3 Encapsulation of nile red or pyrene using mixed initator polydendrons

Stock solutions of nile red in THF at 0.2 mg ml and pyrene in THF at 0.5 mg/ml were made. In a typical experiment the desired amount of nile red or pyrene was added to a vial using a pipette (e.g for a stock solution at 0.2 mg ml, 100 μ! would be used if 0.02 mg was required). The vial was left in the fumecupboard for -20 min to allow evaporation of the THF. A pre-dissolved sample of polymer in THF (1 ml, 5 mg/ml) was added to the vial. The vial was shaken gently to allow dissolution of the fluorescent molecule in the THF containing polymer. Once the desired amount of polymer and fluorescent molecule was dissolved in the 1 m! of THF, this was added quickly to a vial of water (5 ml) stirring at 30 °C. The solvent was allowed to evaporate in a fume cupboard overnight, giving a final concentration of 1 mg ml polymer in water. The nanoparticles formed were analysed by dynamic light scattering (DLS) and fluorimetry.

Table 5 shows data for polymer nanoparticles with a final concentration of 1 mg ml polymer with 0.1 w/w% nile red or pyrene encapsulated (1 . g l)

Tabl - Fluorimetry of nanoparticles with nile red and pyrene encapsulated

G2 DBOP 750 PEG

100 0 pHPMASO 0.8 702.1693 1.42

90 10 pHPMASO 0.8 625.9234 1.4458

75 25 pHPMASO 0.8 574.7425 1.4666

50 50 pHPMASO 0.8 548.357 1.4685

25 75 pHPMASO 0.8 243.2502 1.479

10 90 pHPMASO 0.8 404.1 123 1.5208

0 100 pHPMASO 0,8 285.757 1.5315 1 00 pI-fPMASO 0.95 226.2446

6, Pharmacology

I, Materials & Methods L Materials

Dulbecco's Modified Eagles Medium (DMEM), Hanks buffered saline solution (HBSS), Trypsin-EDTA, bovine serum albumin (BSA), Nile red, 3-(4,5- Dimet!iy1i-hiazol-2-yl)-2,5— diphenylteirazolium bromide (MTT reagent), acetonitrii (ACN) and all general laboratory reagents were purchased from Sigma (Poole, UK), Foetal bovine serum (FBS) was purchased from Gibco (Paisley, UK). The Cel!Titer- G o® Luminescent Cell Viability Assay kit was from Promega (UK). The 24-well HTS transweil plates were obtained from Coming (New York, USA). The 96-well black walled, flat bottomed plates were from Sterilin (^"Newport, UK).

1 A Rt ime cell cuit re/ceii mai teMsnee

Caco-2 ceils were purchased from American. Type Culture Collection (ATCC, USA) and maintained in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 15% filtered sterile foetal bovine serum. Cells were incubated at 37°C and 5% CO2 and were routinely sub-cultured every 4 days when 90% confluent. Cell count and viability was determined using a Countess automated cell counter (Inviirogen).

L2 Cytotoxicity

Caco-2 cells were seeded at a density of 1 ,0 x 10⁴ cells / 100 μΐ in DMEM

supplemented with 15% FBS into each well of a 96 well plate (Nunclon, Denmark) and incubated at 37°C and 5%€·<¼. Cells from 4 separate flasks of biological replicates of each cell type were used (Nl -4) to improve statistical power. Media was then aspirated from column 1 and replaced with media containing each polydendron or aqueous Nile Red solution at an equivalent 1 μΜ Nile Red concentration then d ilnted 1 : 1 in media across the plate up to column 11 , Column 12 served as a negative control and consisted of media and untreated cells. addition, the plates were incubated for 24 hours or 120 hours at 37°C, 5% C<¾ prior to assessment of cytotoxicity.

1.3 MTT assay

Following incubation of treated plates for 24 h or 120 h, 20 μΐ of 5 rag ml^'1 MTT reagent was added to each, well and incubated tor 2 hours. Subsequently, ICXkiL MTT lysis buffer (50% N-N-Dimethylformamide in water containing 20% SDS, 2.5% glacial acetic acid and 2.5% hydrochloric acid, p^'H 4.7) was added to each well to !yse overnight at 37°C\ 5% CO2. Following incubation the absorbance of each well was read using a Tecan Genesis plate reader at 560nm (Tecan Magellan, Austria).

1.4 ATP assay

Following incubation of treated plates for 24 h or 120 h, cells were equilibrated to room temperature for approximately 30 minutes. All but 20 μ! of media, was removed from each well and 20 μί CellTiter-Gio®(Promega, UK) reagent was added. All reagents were made fresh and in accordance with the manufacturer's instructions. Plates were put on an orbital shaker for Ϊ0 minutes to mix contents and allow for stabilisation of luminescence signal. Luminescence was then measured using a Tecan Genios plate reader (Tecan Magellan, Austria).

2. Tra&seeliuiar permeability of Nik Red ac oss Caco-2 monola ers

2,1 Setting up and t eatin tr s? swell plates

Transwells were seeded with 3.5 x 10⁴ cells per well and propagated to a monolayer over a 21 day period, during which media in the apical and basolateral wells was changed every other day. Trans-epithelial electrical resistance (TEER) values were monitored until they were >I3G0fl 1 μΜ of Nile Red po!ydendron or 1 μΜ aqueous Nile Red was added to the apical chamber of 4 wells and the basolateral chamber of 4 wells to quantify transport in both Apical to Basolateral (A>B) and Basolateral to Apical (B>A) direction and sampled on an hourly basis over a 4 h time period.

Apparent permeability coefficient was then determined by the amount of compound transported over time using the equation:

Fapp - άρ/άή /AQi) where (dQ/dt) is the amount per time (nmol. see^"'). A is the surface area of the filter and Co is the starting concentration of the donor chamber (1 μ.Μ).

23 Extraction mid q utifisMk of Nik Red

100 μ! of each collected sample was mixed with 900 μΐ acetone, vortexed, sonicated for 6 minutes and centrifuged at 13300 rpm for 3 minutes. The supernatant was completely dried in a vacuum centrifuge at 30°C until the dry solid sample was left. This was reconstituted in 150 μ! acetonitrile, transferred to a 6-well black walled, flat bottomed plate and measured for fluorescence intensity excitation wavelength 480 run, emission wavelength 560 am using a Tecan Genios plate reader (Teean Magellan, Austria).

3_* Results 3.1 Cytotoxicity - MTT assays

Following 24 hour incubation of Caco-2 cells with each polydendron, analysis of cytotoxicity by MTT assay (Figure 6) showed that aqueous Nile Red and each polydendron did not affect metabolic turnover of Caco-2 cells compared to untreated cells at the range of concentrations investigated. It can be inferred that metabolic turnover correlates to cell viability in which case each material was not cytotoxic.

Figure 6: MTT assay of Caco-2 cells following 24 hour incubation with aqueous Nile Red and each polydendron. A = aqueous Nile Red, EC50 1 .160. B = 0: 100, EC₅₀ 2.509. C = 10:90, EC_5G 1.410. D - 25:75, EC₅₀ 1.567. E - 50:50, EC₅₀ 1.083. F = 75:25, ECso 1 .565, G = 90: 10, EC₅₀ 1 .607. H = 100:0, EC₅₀ 2.678.

Following 120 hour incubation of Caco-2 cells with each polydendron, analysis of cytotoxicity by MTT assay (Figure 7) showed that aqueous NR and each

polydendron at the range of concentrations investigated did not affect the viability of

Caco-2 cells. Figure 7: MTT assay of Caco-2 cells following 120 hour incubation with aqueous Nile Red and each polydendron. A - aqueous Ni!e Red, EC5.3 No ECso. B ^~ 0: 100, EC50 1.528. C = 10:90, EC₅₀ No EC₅₀. D - 25:75, EC50 6.166. E = 50:50, EC50 0.7856. F - 75:25, EC₅₀ No EC50, G === 90:10, EC₅₀ 0.2176. H ^» 100:0, EC₅₀ No

EC

3.3 ATF assay

Following 24 hour incubation of Caco-2 ceils with each polydendron, analysis of cytotoxicity by ATP assay using a CellTiter-Glo® kit (Promega, UK) (Figure 8) indicated that ATP presence was not affected in cells treated with aqueous Nile Red solution and polydendron formulated Nile Red at the range of concentrations investigated compared to untreated cells. It can be inferred that the presence of ATP correlates to cell viability in which case each material was not cytotoxic.

Figure 8: ATP assay of Caco-2 cells following 24 hour incubation with aqueous Nile Red and each polydendron. A = aqueous Nile Red, EC50 1.946. B = 0:100, EC50 2.855. C = 10:90, EC50 No EC₅₀. » - 25:75, EC₅₀ No EC₅₀. E = 50:50, EC₅₀ No ECso. F - 75:25, EC 50 No EC50, G - 90:10, EC₅₀ 2.848. H - 100:0, EC_S 0.1961.

Following 120 hour incubation of Caco-2 cells with each polydendron, analysis of cytotoxicity by ATP assay using a CellTiter-Glo® kit (Promega, UK) (Figure 9) indicated viability was not affected in cells treated with aqueous Nile Red solution and each polydendron material at the range of concentrations investigated compared to untreated cells.

Figure 9: ATP assay of Caco-2 cells following 120 hour incubation with aqueous Nile Red and each polydendron. A ^:::: aqueous Nile Red, EC50 No EC50. B -· 0:100, EC₅.3 N0 EC_5C. C - 10:90, ECso 3.168. P - 25:75, EC_S0 2.565. E - 50:50, EC₅₀ No ECso. F = 75:25, EC50 3.032, G ^« 90:10, ECso No EC₅₀. H = 100:0, EC₅₀ No EC₅Q.

4. Trass see!Mar permeability of selected Nile Red polyde&ctroR materials ac oss Caco-2 cell monolayers. Transcellular permeability of Nile Red through Caco-2 ceil monolayers (to model the intestinal epithelium) was significantly higher in the apical to basoiateral (A>B, gut to blood) direction for the polydendron preparation 10G2:90PEG compared to an aqueous solution of Nile Red (Figure 10 A&B). All the polydendron materials

5 produced a greater apical to basoiateral (A>B, gin to blood), basoiateral to apical (B>A, blood to gut) ratio than an aqueous preparation of Nile Red following 1 hour incubation (Table 1, Figure 10 C). A statistically significant correlation (P^:::<0.05) between the ratio of dendron and PEG used in the polydendron formulation and the ratio of apical to basoiateral (A>B, gut to blood), basoiateral to apical (B>A, blood to

10 gut) movement of Nile Red across the Caco-2 monolayer was observed (Figure 10 C).

Figare 10. (A&B) Transcellular permeability across Caco2 cell monolayers of polydendron formulated Nile Red relative to an aqueous solution of Nile Red. Data are given as the mean of experiments conducted in biological triplicate. (C)

Correlation between polydendron formulation and the ratio of Nile Red transported (A>B B>A) across Caeo2 cell monolayers (Γ 0.784). Data were normally distributed statistical analysis was conducted using a Pearson correlation (P=<0.05) a two-tailed P value was used to reduce the chance of a type Ϊ error.

Table 1. Apparent permeability (Papp) of Nile Red po!ydendrons and aqueous Nile Red across Caco2 cell monolayers following 1 hour incubation. Data are given as the mean of experiments conducted in biological triplicate.

tq eous Nile Red x

7, Example of nanoprecipitation to encapsulate inorganic magnetic

nanoparticles

5

Folydendron (G2:2 FEG(50:50HpHPMA_So~EGDMA₀._s) was dissolved In THF for a minimum of 6 hours. Once foily dissolved the polymer in THF (0.2 mi, 25 mg mi) was mixed with F j0₄ 10 nm particles in THF (0,5 ml, 5 mg ml) and this mixture of polymer and FesO was added quickly to a vial of water (1 ml) stirring at 30 °C. The0 solvent was allowed to evaporate overnight in a fume cupboard to give a final

concentration of 5 mg/ml polymer, 2.5 mg/ml Fes0 in water. The nanoparticles formed were analysed by dynamic light scattering (DLS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). 5 SEM imaging showed spherical nanoparticles of size range varying from

approximately 150 to 250 nm while TEM imaging showed the majority of nanoparticles to have encapsulated Fes04 with no free Fe.¾04 observed.

DLS (2.5 mg ml in water) determined the Z-Ave hydrodynamic diameter to be 182 0 nm with PDI to be 0.01 , In the presence of a magnetic field (i.e. with a magnetic suspended above, just touching the surface of the dispersion) DLS measurements showed a 50% reduction in derived count rate after 12 hours and a 40% reduction in derived count rate after 8 hours, with Z-Ave diameter remaining constant throughout.

The reduction in derived count rate is intrinsic to a decrease in concentration of 5 nanoparticles within the dispersion and demonstrates the effect of the magnetic field on directing the behaviour of the nanoprecipitate. In the absence of a magnetic field there is no drop in derived count rate.

Claims

A method of preparing a non-gelled branched vinyl polymer scaffold carrying dendrons, comprising the living or controlled polymerization of a monofunciional vinyl monomer and a di functional vinyl monomer, using a dendron initiator and at least one further initiator.

A method as claimed in claim 1 wherein the living polymerization is ATRP.

A method as claimed in claim 1 or claim 2 wherein the molar ratio of Afunctional vinyl monomer to initiators is less than 1 .

A method as claimed in any preceding claim wherein the further initiator is selected from or comprises one or more of the following: a small molecule, a drug, an active pharmaceutical ingredient, a polymer, a peptide, a sugar, a dendron, a moiety which carries or can carry a drug, an anionic functional group, a cationic functional group, a moiety which enhances solubility (for example, of the polydendron within aqueous systems, or of a drag or other carried material), a moiety which, prolongs residence time within the body, a moiety which enhances stability of a drug or other active material, a moiety which reduces macrophage uptake, a moiety which enhances controlled release, a moiety which enhances drug transport, or a moiety winch enhances drug targeting.

A method as claimed in any preceding claim wherein the further initiator comprises a PEG group.

A method as claimed in any preceding claim wherein the dendron initiator comprises a generation 1 dendron.

A method as claimed in claim 6 wherein the first generation branches are identical.

8. A method as claimed in any preceding claim wherein the dendron initiator comprises a generation 2 dendron.

9. A method as claimed in claim 8 wherein the second generation branches are

identical.

10. A method as claimed in any preceding cSaim wherein one or more of the initiators comprises a functional group allowing post-fonctionalization, i ! . A method as claimed in any preceding claim followed by nanoprecipitarion to form nanopartic!es,

12, A method as claimed in any preceding claim wherein the ono&nctional vinyl

monomer and/or the difunetional vinyl monomer comprise a methaerylate,

1.3. A product obtainable by the method of any preceding claim.

14. A non-gelled branched vinyl polymer scaffold carrying one type of dendron moiety and a furdrer moiety.

15. A product as claimed in claim 14 which is an atom transfer radical polymerized

material .

16. A product as claimed in claim 14 or claim 15 wherein the further moiety is selected from one or more of the following: a small molecule, a drug, an active

pharmaceutical ingredient, a polymer, a peptide, a sugar, a dendron, a moiety which carries or can carry a drug, an anionic functional group, a cationic functional group, a moiety which enhances solubility (for example, of the poiydendron within aqueous systems, or of a drug or other carried material), a moiety which prolongs residence time within the body, a moiety which enhances stability of a drug or other active material, a moiety which reduces macrophage uptake, a moiety which enhances controlled release, a moiety which enhances drug transport, or a moiety which enhances drug targeting,

17. A product as claimed in any of claims 14 to 1 6 wherein the further moiety comprises a PEG group.

I S, A product as claimed in any of claims 14 to 17 wherein the dendron initiator

comprises a generation 1 dendron.

19. A product as claimed in claim 18 wherein the first generation branches are identical.

20. A product as claimed in any of claims 14 to 19 wherein the dendron initiator

comprises a generation 2 dendron.

21 . A product as claimed in claim 20 wherein the second generation branches are

identical.

22. A product as claimed in any of claims 14 to 21 wherein one or more of the initiators comprises a functional group allowing post- unctionalization.

23. A nanopariicle comprising a product as claimed in any of claims 14 to 22.

24. A pharmaceutical composition comprising a product as claimed in any of claims 13 to 23 and a pharmaceutically acceptable diluent.

25. A pharmaceutical composition as claimed in claim 24 which is orally administrab!e,

26. A pharmaceutical composition as claimed in claim 24 which is parenteraliy

adrninistrab!e.

27. A pharmaceutical composition as claimed in claim 24 which is topically administrabk.

28, A pharmaceutical composition as claimed in claim 24 which is administrate to the eye.

29, A product as claimed in any of claims 13 to 23 for use in therapy.

30. A product as claimed in any of claims 13 to 83 for use as an oraHy, topically or parenterally administered medicament.

31. A method of treatment comprising administration of a product as claimed in any of claims 13 to 28 to a patient in need thereof.